SPECIAL EDITION

CLEANING
VALIDATION

Published by:
CLEANING VALIDATIoN
Validation Case Study: Erroneous Negative Cleaning Validation Results | IVT ...................................................... 1

Experimental Parameters for Small-scale Cleaning Characterization

Part I: Dilution of Process Fluids During Cleaning | IVT ....................................................................................... 7

Experimental Parameters for Small-Scale Cleaning Characterization.

Part II: Effect of Fluid Velocity on the Kinetics of Cleaning | IVT ........................................................................ 11

Methodology for Assessing Product Inactivation during Cleaning

Part I: Experimental Approach and Analytical Methods | IVT ............................................................................. 16

Methodology for Assessing Product Inactivation During Cleaning

Part II: Setting Acceptance Limits of Biopharmaceutical Product Carryover for Equipment Cleaning | IVT ........... 20

Aseptic Transfer Risk Assessment: A Case Study | IVT ...................................................................................... 27

People in Cleanrooms: Understanding and Monitoring the Personnel Factor | IVT .............................................. 32

PAT: Using PAT to Support the Transition from Cleaning Process Validation to Continued Cleaning Process
Verification | IVT ............................................................................................................................................. 39

Translating Laboratory-Developed Visual Residue Limits to Process Area Applications | IVT .............................. 45

New Perspectives on Cleaning: Cleaning Validation of Multiproduct EquipmentAcceptance Limits for Inactivated
Product | IVT ................................................................................................................................................... 51

Biopharmaceutical Cleaning Validation: Acceptance Limits for Inactivated Product Based on Gelatin as a
Reference Impurity | IVT .................................................................................................................................. 60

Multiproduct Cleaning Validation: Acceptance Limits for the Carryover of Inactivated API Part IThe Comparable
Quality Approach ............................................................................................................................................. 67

Validation of a Cleaning Process for Medical Devices | IVT ............................................................................... 72

Ensuring Sterility: Autoclaves, Wet Loads, and Sterility Failures | IVT ............................................................... 79
2015 Advanstar Communications Inc. All rights reserved.
Reproduction in whole or part is prohibited without prior written permission of the publisher.
CLVQ4005
 Paul L. Pluta

Validation Case Study:

Erroneous Negative Cleaning
Validation Results | IVT
Paul L. Pluta, Ph.D.

Validation Case Studies provides a forum for validation practitioners to share

information about actual validation experiences. Previous discussions ad-
dressed a wide range of activities. Previous case study titles discussed in this
series include the following:

1. Visual Observations, Journal of Validation technology ( JVT), Volume

16, #1.
2. Equipment Qualification, JVT, Volume 16, #1.
3. Identical mixing Tanks, JVT, Volume 16, #3.
4. Cleaning HPLC Peaks, JVT, Volume 16, #4.
5. Documentation Practices, JVT, Volume 17, #1.
6. Yield, JVT, Volume 17, #2.
7. Like-for-Like Changes, JVT, Volume 17, #2.

Readers are invited to participate and contribute manuscripts for this

series -- we encourage sharing successful practices with others. Please contact
journal editor-in-chief Paul Pluta at paul.pluta@comcast.net or content spe-
cialist Dustin Henderson at dustin.henderson@cbinet.com with comments or
submissions for publication.

ABSTRACT
This case study describes a cleaning validation event in which failing
results for API residue from a small molecule extended release tablet
dosage form were observed. The initial two lots in the cleaning valida-
tion were successful. The third lot failed acceptable residue limits.
Investigation of the failure comprised cleaning process development
and performance; residue sampling, sample handling, sample analy-
sis, and evaluation of the analytical method. Investigation of this
event initially involved interviews of relevant personnel and reviews
of associated documentation. Two areas were identified for further
evaluation residue sampling and the cleaning process. Regarding
sampling, a newly trained technician, working alone, sampled the first
two acceptable lots, while an experienced technician working with a
colleague sampled the third failing lot. Evaporation of sampling sol-
vent occurred causing residue to be insufficiently recovered from the
equipment surface resulting in erroneous false negative test results.
Regarding the cleaning process, manufacturing operators commented
that the new extended release formulation was more difficult to clean
than the original immediate release formulation although the same
cleaning procedure was utilized for both products. Evaluation of the
cleaning process indicated that the process parameters were not opti-
mal to clean the new extended release product. An improved cleaning
process with increased cleaning agent concentration, increased clean-
ing time, and higher temperatures was developed, implemented, and
ultimately validated.

Special edition: Cleaning Validation 1

Paul L. Pluta
Cleaning validation sampling personnel must
have good technical understanding of their work,
and must know the technical reasons for the pro-
cedures they perform, and potential problems if
procedures are not correctly performed. Sampling
personnel training for cleaning validation should
include a quantitative demonstration of acceptable
cleaning by means of analytical testing. Training
exercises must also include worst-case sampling
such as with volatile solvents, multiple equipment,
and other potential variations in sampling. SOPs
must be carefully written to describe potential
problems and include performance constraints to
minimize variation and risks. There is an inherent
danger when variation is not deliberately intro-
duced into a validation project material variation,
manufacturing operator variation, and in this case
study, sampling personnel variation. Sampling by
two different technicians enabled erroneous results
to be discovered. Regarding the cleaning process,
inactive ingredients in a formulation may have very
significant effects on cleaning processes. Cleaning
of residues does not depend solely on the proper-
ties of the API.
INTRODUCTION
The compliance event involved cleaning valida-
tion for cleaning of residue from a new small
molecule extended release tablet dosage form.
The active ingredient in the tablet was a potent
drug dosage of the active ingredient was low
and cleaning was expected to be successful. The
site already had a successful history of cleaning
a marketed immediate-release tablet containing
a lower dose of the same active drug. No changes
in the cleaning method were required for the new
product. After cleaning, the target residue level
was below visually clean and required the resi-
due level to be determined by swab sampling and
chemical analysis. Three lots were required for
cleaning validation.
The following are discussed:
Compliance event. A description of the cleaning
validation event
Investigation. Interviews and actions conducted
to investigate the event
Discussion. Key information, activities, and anal-
ysis
CAPA. Actions and improvements implemented
in the cleaning process, sampling process for
cleaning validation, and training of sampling per-
sonnel
Cleaning validation of modified cleaning process.
Implementation of the new cleaning process and
subsequent validation.
2 Special edition: Cleaning Validation
BACKGROUND
The process of validation typically comprise the fol-
lowing sequence of activities:
1. Change desired. An equipment or process
change is needed or required. This may be a
necessary change or a desirable improvement.
2. Development work. Appropriate Stage 1 devel-
opment work is completed in support of the
change.
3. Validation plan. A formal request to initiate the
validation process is submitted to the valida-
tion approval committee (VAC). Development
reports may be included in the request in sup-
port of the change. The change request includes
a proposed level of work to confirm the accept-
ability of the change. The level of work is based
on risk to the patient and to the organization.
The VAC approves the change request. The
approved change request document is stored in
the validation library.
4. Protocol. A protocol is written specifying
detailed sampling and testing to confirm the
acceptability of the change. The VAC approves
the protocol. The approved protocol is stored in
the validation library.
5. Validation work. Stage 2 PPQ validation work
is performed according to the protocol. Sam-
pling and testing are completed. Data and other
results are generated and recorded.
6. Validation report. A report containing all
test results with discussion and conclusions
is prepared and submitted to the VAC for
approval. The report is approved and the pro-
cess or equipment change is implemented. The
approved validation report is stored in the vali-
dation library.
7. Validation closure. If no other work is needed,
the validation project initiated by the change
request is closed.
8. Continued verification. Stage 3 post-validation
monitoring confirming acceptability of the
change continues throughout the product / pro-
cess lifecycle.
The issue addressed in this case study occurs
in #5 and #6 above. The actual work conducted to
confirm the acceptability of the validation project is
performed by technical people. In this case study,
samples were removed and tested for residue content.
VALIDATION EVENT
A small molecule pharmaceutical company conducted
initial cleaning validation on a new extended release
tablet product containing a water-insoluble API as
active ingredient. The new product was a line exten-

sion -- an extended release formulation of a marketed
immediate-release tablet product. The new product
contained a polymeric matrix to enable prolonged
release and once-daily dosage to patients.
The cleaning validation exercise was expected
to be successful. Although the product contained a
highly potent active drug which required low residue
levels on cleaned equipment, the company had ex-
tensive experience with the cleaning procedure over
several years. The original immediate-release product
cleaning was relatively easy and had a long success-
ful history of performance. Several previous cleaning
validations had been successfully accomplished. The
analytical method for residual API from swab samples
was easily performed and very reliable.
Sampling of three lots of new product was planned
for cleaning validation. The manufacturing process
comprised several unit operations. Sampling of unit
operations for cleaning validation was performed on
multiple days for each lot. The first lot was manu-
factured and cleaning completed on all equipment.
Equipment was visually clean. Swab sampling was
done by the sampling technician. Cleaning validation
analytical test data indicated no active drug present
in all swab samples all acceptable results. A second
lot was manufactured. Cleaning was completed. Swab
sampling was done. Cleaning validation analytical
test data again indicated no levels of residual drug
in all swab samples. A third lot was manufactured.
Cleaning was completed. Swab sampling was done.
Cleaning validation analytical test data indicated
extremely high residue levels significantly above
the required acceptance criteria. Test results on the
third lot indicated a significant failure of the cleaning
process.
This event prompted multiple questions to be investi-
gated and answered.
1. Cleaning process performance. Did manufac-
turing personnel correctly perform the cleaning
process in the third (failing) lot? Which opera-
tor cleaned the equipment? Were manufactur-
ing personnel adequately trained in the cleaning
procedure? What was past history with use of
this cleaning process? Were repeat cleanings
required in past cleaning? Were deviations
required?
2. Cleaning process development. How were the
cleaning process parameters developed? What
was the history of this method with the imme-
diate release product? We any changes made for
cleaning the extended release product?
3. Sampling. Did the sampling technician cor-
rectly sample the recommended equipment
surfaces? Were sampling personnel adequately
trained?
Paul L. Pluta
4. Residue samples. Was the integrity of residue
samples adequately protected during transport
to the lab? Could samples have become contam-
inated causing the test failures? Were samples
correctly and quickly transferred to the lab for
analysis? Were samples handled during trans-
port and storage according to procedures? Were
samples exposed to high temperatures during
transport and storage?
5. Analytical laboratory. Was the analysis correctly
performed? Which technician performed the
analysis? Were laboratory technicians adequate-
ly trained? Was analytical equipment qualified
for use for the API analysis? Was system suit-
ability below required limits?
6. Analytical R&D. Were there any problems
with the analytical method? Was the analytical
method correctly developed? Was the analytical
method validated?
INVESTIGATION
Investigation of this compliance event initially in-
volved interviews of relevant personnel and reviews
of associated documentation. Personnel related to the
compliance event included manufacturing personnel,
QC personnel, cleaning sampling technicians, and
the technical personnel responsible for product for-
mulation and process, cleaning method development,
technical support, and analytical testing. There were
many details and variables that needed to be inves-
tigated and/or confirmed. Personnel from all groups
interacted to address the above issues.
Documentation reviews included manufacturing
documentation, cleaning documentation, equipment
inspection records, laboratory records, analytical
method development reports, validation reports, and
other records. All applicable manufacturing SOPs
and analytical SOPs were reviewed.
Cleaning Process Performance
Manufacturing personnel correctly performed the
cleaning process in all three lots. Cleaning procedure
documentation for all lots was reviewed and found to
be perfectly executed. No deviations were issued. Dif-
ferent operators executed cleaning of multiple equip-
ment in the validation lots. An experienced operator
executed cleaning in the third (failing) lot. Training
records for all operators were reviewed and found to
be acceptable. Operators commented that the new
extended release product was more difficult to clean
than the original immediate release product. The
extended release polymer made removal of the prod-
uct residue more difficult that was typical with the
original immediate-release product. This observation
reflected operator experience with manual cleaning
of small parts. Product was able to be removed from
Special edition: Cleaning Validation 3
Paul L. Pluta
equipment surfaces and yield visually-clean surfaces.
No repeat cleanings were required. No deviations
were issued.
Cleaning Process Development
The cleaning process for the immediate release
product had been previously developed. An alkaline
cleaning agent that was used on several other prod-
ucts in the plant was used. Because the API in the
new extended release product was the same as in the
immediate release product, no changes were made
to the cleaning method. Technical personnel were
unaware of any difficulty in cleaning the extended
release product.
Sampling
Two different sampling technicians performed sam-
pling in the three lots. A newly-trained technician
sampled lots #1 and #2, both of which had accept-
able low residue levels. The newly-trained techni-
cian worked alone to accomplish sampling since
no other sampling technicians were available. Lot
#3 was sampled by an experienced technician. The
experienced technician worded with a colleague who
helped sample the recommended equipment surfaces
and complete required documentation. Both sam-
pling personnel were adequately trained as evidenced
by training documentation.
Residue samples
Residue samples were packaged in protective wrap-
ping for transfer to the lab. Samples were immediately
closed and not contaminated. Transport to the lab
was rapid and without exposure to unusual environ-
mental conditions or excessive heat.
Analytical laboratory
The laboratory analysis was correctly performed.
Experienced technicians performed the analysis.
All technicians were adequately trained. Analytical
equipment was qualified for use for the API analysis.
Lab documentation indicated acceptable execution of
the analytical procedure.
Analytical R&D
The same analytical method was used for the original
irradiate-release product and for the new extended-
release product. Analytical R&D verified that the test
method performed acceptably for the new product.
There were no problems with the analytical method.
The analytical method was validated.
DISCUSSION
Interviews and discussion of the above questions did
not clearly indicate an obvious cause for the prob-
lem. Manufacturing personnel confirmed that they
4 Special edition: Cleaning Validation
performed cleaning as required by procedure. Equip-
ment was cleaned by automated methods wherever
possible. All associated small parts were manually
cleaned was cleaned according to procedure. The
manufacturing supervisor verified that procedures
were followed and that the equipment was visu-
ally clean. Quality unit personnel who inspected
the equipment also verified that all equipment and
small parts were visually clean. All inspections were
conducted after the equipment was dry. Samples
were transported to the lab quickly and according to
procedure. Samples were also quickly stored in the
laboratory upon receipt and under specified security
conditions. Laboratory personnel confirmed accept-
able performance of analytical procedures. Analytical
standards over a range of concentrations tested along
with the actual cleaning validation samples yielded
accurate results. Analytical R&D scientists confirmed
acceptable performance of the validated test method.
Two areas were identified for further investigation.
These included:
1. Swab sampling. A newly trained technician,
working alone, sampled the first two acceptable
lots. All samples in these lots were acceptable.
An experienced technician, working with a
colleague, sampled the third failing. Was some-
thing different about the third lot, or was the
failing data due to the difference in sampling
personnel?
2. Cleaning process. Manufacturing operators
commented that the new extended release for-
mulation was more difficult to clean than the
original immediate release formulation. The
same cleaning procedure was utilized for both
products.
Swab Samplingy
Swab sampling for the three lots was done by two
different sampling technicians. The first two lots were
sampled by a newly-trained person. Data for these
lots indicated minimal or no residual soil acceptable
results. The third lot with failing data was sampled
by an experienced technician who worked with a
colleague.
The sampling method required wetting of the
swab with organic solvent to dissolve residue from
the equipment surface. The new technician did all
sampling alone. The experienced technician per-
formed sampling with a colleague to accomplish the
sampling procedure in minimum time. She explained
the necessity of the rapid sampling technique because
evaporation of the sampling solvent must be mini-
mized. The new technician was not aware of the time
limitation in sampling. Although not conclusively
proven, it was suspected that evaporation of solvent

occurred causing residue to not be adequately recov-
ered from the equipment surface. The new technician
worked slowly and carefully, and completed all neces-
sary steps. However, the time required for perfor-
mance, especially since she worked alone, may have
caused residue recovery to be incomplete or minimal.
The analytical lab confirmed that if sufficient solvent
was not present on the swab, residue recovery would
be unsuccessful.
Cleaning Process
Technical personnel responsible for the cleaning
process had no previous experience with the clean-
ing method. The cleaning method for the original
product had been established many years ago and
never required new technical evaluation. Manufactur-
ing management decided to use the well-established
cleaning method without involvement of technical
personnel. Managements rationale was that since the
API in the original product had been reliably cleaned
for many years, there was no need to evaluate the
cleaning process for the new product. Technical per-
sonnel had not been requested to evaluate the clean-
ing process used in the failed cleaning validation.
In light of the cleaning failure, technical personnel
recommended laboratory studies to evaluate available
cleaning agents, cleaning process parameters, and
related factors in a systematic way. Evaluation of the
cleaning process indicated that the process param-
eters were insufficient to clean the new product. The
polymeric matrix in the new product (methylcellulose
mixture) was much more difficult to clean than the
original immediate release product. Technical per-
sonnel conducted studies to establish new cleaning
process parameters suitable for the extended release
product. A new cleaning method with increased
cleaning agent concentration, increased cleaning
time, and higher temperatures was developed.
CORRECTIVE ACTION / PREVENTIVE ACTION (CAPA)
Two CAPA activities corrected the problems expe-
rienced in the original cleaning validation. These
involved new training of swab sampling personnel
and a modified cleaning process for the extended
release product.
Swab Sampling Training
Personnel who perform cleaning residue sampling
using swabs wetted with volatile solvents were taught
the importance of rapidly performing swab sampling.
Many of the swab sampling technicians did not have
a technical background and did not understand
solvent volatility and the consequences for swab
sampling. Studies confirmed that the new techni-
cian, who worked alone in the sampling activity, did
not perform swab sampling quickly. When sampling
Paul L. Pluta
was not performed quickly, solvent evaporated and
surface residue was not able to be dissolved. Analyti-
cal results on evaporated swab samples indicated
extremely low or no levels of residue which errone-
ously passed cleaning validation acceptance criteria
a false negative due to solvent evaporation.
Future training of swab sampling technicians
included new test procedures to require rapid
performance of sampling procedures. The previous
qualification test did not utilize a volatile solvent and
did not require rapid performance. The new qualifi-
cation test required technicians to demonstrate rapid
sampling in order to become a qualified sampling
technician. Sampling teams (two technicians) were
required when volatile solvents were used in sam-
pling. Technicians were required to quantitatively
recover residue in training to be qualified for residue
sampling. Training was repeated on an annual basis.
SOPs describing cleaning sampling methods using
volatile solvents were strengthened to require rapid
sampling and working in teams. The combined em-
phasis of new training and new procedures that both
underscored the risks and potential variation in resi-
due sampling strongly addressed the issues described
in this case study.
Modified Cleaning Process for Extended Release
product
Technical personnel evaluated the cleaning process
and determined that process parameters were not op-
timal to reliably clean process residues. The cleaning
agent concentration was increased, the temperature
was increased, and the cleaning time was increased
in the new procedure. These parameters enhanced
the cleaning process to more effectively and more ef-
ficiently remove the polymeric residue.
CLEANING VALIDATION OF MODIFIED CLEANING
PROCESS
The new cleaning process was implemented. Manu-
facturing operators confirmed that new cleaning
process parameters significantly improved the clean-
ing process. Three product lots were manufactured.
Cleaning was performed on required equipment in
three lots. Worst-case locations on equipment were
swab sampled by two-person teams of sampling per-
sonnel. Two-person teams ensured minimal solvent
evaporation and rapid sampling procedures. All test
results passed the acceptance criteria.
SUMMARY AND FINAL THOUGHTS
A case study describing a compliance event in which
erroneous false negative analytical data was generated
in cleaning validation. These data caused a mistaken
conclusion that a cleaning process for a new modi-
fied release dosage form was acceptable. The cause
Special edition: Cleaning Validation 5
Paul L. Pluta
of the problem was not easily determined all test
data were acceptable. Initial investigation of potential
problem areas indicated that everything was done
according to procedure nothing was done incor-
rectly. It was ultimately determined that the sampling
process for product residue was not sufficiently con-
trolled, and that the equipment cleaning process was
not adequate for the modified release formulation.
The sampling error, i.e., loss of solvent in sampling,
had a major effect on cleaning validation. The sam-
pling technician did not understand the importance
of working quickly to minimize solvent loss. This
lack of understanding resulted in a false negative test
result and an erroneous conclusion that the cleaning
process was acceptable. Fortunately the error was
discovered when a different technician correctly and
rapidly sampled the equipment surfaces. The com-
bined emphasis of new training and new procedures
that both emphasized the risks and potential varia-
tion of sampling strongly addressed the sampling
issues described in this case study. Observations by
manufacturing personnel caused the cleaning process
to be evaluated by technical personnel, and a new
cleaning process with optimized process parameters
was developed. The new cleaning process was ulti-
mately validated.
Lessons Learned
Several important lessons may be learned from this
case study.
Sampling personnel understanding of sam-
pling process and training. Sampling person-
nel must have good technical understanding
of their work. They must know the reasons for
6 Special edition: Cleaning Validation
the procedures they perform. They must know
potential problems if procedures are not correctly
performed.
New procedures. SOPs describing cleaning
sampling methods using volatile solvents were
strengthened to require rapid sampling and
working in teams. Time constraints were added
to all affected procedures. SOPs must be carefully
written to identify potential risks and minimize
variation.
Sampling personnel training. Training of
cleaning validation sampling technicians is a
critical activity. Training exercises must include
a quantitative demonstration of acceptable
cleaning by means of analytical testing. Training
exercises must also include worst-case sampling
such as with volatile solvents, multiple sampling
equipment, and other potential variations used
in sampling. Retaining of technicians at some
defined and reasonable frequency should be
considered.
Inactive ingredients effects on cleaning. Inac-
tive ingredients may have very significant effects
on cleaning processes. Cleaning of residues
does not depend solely on the properties of the
API. Formulation ingredients may significantly
affect the cleaning process. In this case study,
an extended release polymer in the formulation
caused difficulty in the cleaning process. Inactive
ingredients such as dyes and flavors may also
greatly influence cleaning, and may actually be
the most difficult ingredients to clean in a formu-
lation. All components in a formulation must be
considered when developing a cleaning process.
 Rizwan Sharnez

Experimental Parameters
for Small-scale Cleaning
Characterization Part I:
Dilution of Process Fluids
During Cleaning | IVT
Rizwan Sharnez, Ph.d., Angela to, Arun tholudur, Ph.d.

CLEANING VALIdAtIoN
Methodologies for estimating experimental parameters for small-scale cleaning
characterization studies are described in this series: dilution of process fluids (e.g.,
process soil, cleaning solution, or rinse water) during cleaning is discussed in this
paper; worst-case fluid velocity and soil load will be discussed in subsequent parts.
Dilution of the process fluid during cleaning was estimated to be on the order of
1016 for a typical cleaning cycle and 105 for intermediate cleaning steps. These
dilution factors are used to estimate the concentration of impurities in the final
rinse and to simulate worst-case cleaning conditions for cleanability and inactiva-
tion studies.

INtRodUCtIoN
Small-scale cleaning characterization (CLC) studies are used to identify suit-
able cleaning chemistries (1, 2), optimize cleaning parameters and processes
(3, 4), establish cleaning times for manufacturing equipment (5, 6), and
streamline validation requirements for multiproduct equipment (7, 8).
Experimental models for small-scale CLC have been described in the
literature (9-12). In these experiments, the kinetics of soil removal (mass
transfer) from a surface is measured under simulated cleaning conditions. A
critical step in the development of these models is to identify and scale down
the hardest-to-clean (worst-case) location in the equipment (13). Note that
it is not necessary to simulate the entire cleaning process; instead, it is only
necessary to simulate the location within the equipment that is the hardest
to clean. If the process soil can be adequately removed from the worst-case
location, it follows that it can also be adequately removed from the other
locations in the equipment. Further, with this approach, the cleaning times
obtained at small-scale would be indicative of those at full scale assuming
that there is adequate coverage at the surfaces that need to be cleaned.
The scalability of small-scale CLC data depends on the accuracy with
which the experimental parameters are estimated. Experimental parameters
for simulating the worst-case location fall into two categories (14):

Parameters that can be readily determined from process data such as:
o Material of construction and surface smoothness of coupons or parts
used to simulate large-scale equipment
o Post-soiling parameters such as hold time, temperature, and humidity
o Cleaning parameters such as rinse or wash time, temperature of rinse
solvent, and temperature and concentration of cleaning solution.
Parameters that typically need to be determined from first principles
such as:
o Dilution of the process fluid during cleaning

Special edition: Cleaning Validation 7

Rizwan Sharnez

o Velocity of rinse solvent or cleaning solution at the worst-case process soil from the standpoint of drainage.
worst-case location Also, residual volumes obtained with curved surfaces
o Soil load at the worst-case location. (pipes) were significantly greater than that for flat sur-
faces (plates). Consequently, pipes were used to estimate
Residual Process Fluid the worst-case residual volumes.
The volume of residual process fluid (VR) can be esti-
mated from the surface area of the equipment that makes
contact with the process fluid (SA) and the residual
volume of the process fluid per unit surface area (R):

VR = SA R [Equation 1a]

R can be determined experimentally by measuring the

amount of process fluid that remains on a surface after it
is drained. An experimental method for estimating R is
described in the next section.
VR for a CIP circuit can be estimated by dividing the
equipment into the following sections: vessel walls (W);
bottom dish of vessel (D); and associated piping, filter Figure 1: Experimental Setup for Estimating Residual Process Fluid.
housings, and other miscellaneous parts (P). Thus,
The experimentally determined residual volumes for the
VR = SAw Rw + SAD RD + SAP RP [Equation 1b] configurations that were tested are given in Table I.
Note that impellers, filter housings, and other com-
For a tank, the surface area of the sidewall that makes ponents associated with the vessel may contain surfaces
contact with the process fluid is at a range of angles, typically between 5 and 90. The
residual volume for these components may be set to the
SAw = dh [Equation 2] R values for the smaller angle of repose (5) because it
represents a worse case from the standpoint of drainage.
Where d is the diameter of the tank and h is the height
up to which the process fluid wets the sidewall.
The bottom dish surface area (SAD) is estimated from
ASME engineering tables (15). The surface area of piping,
filter housings, and other miscellaneous components
(SAP) is estimated with a 15% overage factor. Thus,

SAP = 0.15 (SAw + SAD) [Equation 3]

Estimation of Residual Process Fluid
The experimental method for estimating the residual
volume of a process fluid (R) is described in this section.
Two flanged stainless steel sections of piping and an end
cap were connected as shown in the Figure. The pipes
were one inch in diameter and four inches in length.
The bottom section of piping was used to model the
continuous nature of surfaces at full scale; it was not part
of the surface area that was used to estimate the residual
volume. The piping assembly was filled with the process
fluid, turned to the appropriate angle (5 or 90), and
then drained by removing the end cap. The amount of
residual process fluid in the top section was measured by
gravimetry.
Residual volumes were estimated for rinse water and
process soil. These fluids were simulated with deion-
ized (DI) water (< 1 S/cm) and a 56% glucose solution
(w/w), respectively. Due to its very high concentration
and viscosity, the glucose solution was representative of a
table I: Residual Volumes per Unit Surface Area (R) for Deionized Water (<1
S/cm) and Process Soil.
dilution of Process Fluid during Cleaning
For a given step in the cleaning process, the dilution of
the process fluid (DS) is determined by the ratio of the
volume of the cleaning solvent or solution (VS) to the
volume of the residual process fluid on the surface that is
being cleaned (VR):
DS = VS / VR [Equation 4]
VS can be obtained from the cleaning cycle param-
eters, and VR can be estimated as described in the previ-
ous section.
Equipment cleaning cycles typically consist of mul-
tiple steps such as a pre-rinse, alkaline wash, post-alka-
line rinse, acidic wash, post-acidic rinse, and final rinse.
The residual process fluid is serially diluted during each
step of the cleaning cycle. The cumulative dilution of the

8 Special edition: Cleaning Validation


process fluid (DC) is the product of the DS values for all
of the subsequent cleaning steps to which the process
fluid is subjected:
[Equation 5]
Where i denotes the ith step and n is the total number
of subsequent cleaning steps.
Note that the above equations do not account for any
change in the volume of the residual process fluid (VR)
due to drying. Thus, this approach is only applicable
when VR is negligible.
Estimation of dilution during CIP
In this section, the dilution of a process fluid is estimated
for a CIP circuit for a 10,000 liter (L) vessel. The vessel
has a diameter (d) of 84 inches (2.14 m), and the process
fluid wets the side wall of the vessel up to a height (h)
of 120 inches (3.04 m). The cleaning steps and solution
volumes for the circuit are given in Table II.
The surface area of the vessel in contact with the pro-
cess fluid is estimated from Equations 2 and 3 and the
engineering table for ASME domed heads (15):
SAW = dh = 20.4 m2
SAD = 4.24 m2
SAP = 0.15 (SAD+SAW) = 3.70 m2
The residual volume per unit area (R) for the above
surfaces is obtained from the R-values for 56% glucose
(representative of the worst-case process soil) in Table
I. The R value for a 90 angle of repose (6.60 mL/m2) is
used to estimate the residual volume for the side walls
(RW), and the R value for a 5 angle of repose (26.5 mL/
m2) is used to estimate the residual volumes for the ves-
sel dish and miscellaneous parts (RD and RP).
The total residual volume of process fluid in the circuit
(VR) is calculated by substituting the above surface areas
and R values into Equation 1b:
VR = SAW RW + SAD RD + SAP RP
= (20.4 m2) (6.60 mL/m2) + (4.24 m2) (26.5 mL/m2) +
(3.7 m2) (26.5 mL/m2)
= 345 mL
The dilution of the process soil for each cleaning step
(DS) and the cumulative dilution (DC) are calculated from
the residual volume of process fluid calculated above and
the volumes of cleaning solution given in Table II. DS and
DC for the pre-rinse and alkaline wash steps are calcu-
lated as follows:
Pre-rinse (PR):
Rizwan Sharnez
DS,PR = VS,PR / VR = 125 L / 0.345 L = 362
DC,PR = DS,PR = 362
Alkaline wash (AW):
DS,AW = VS,AW / VR = 312 L / 0.345 L = 904
DC,AW = DS,PR x DS,AW = 362 x 904 = 3.27 x 105
table II: Dilution of Process Fluid During Cleaning.
Assuming that an impurity has negligible affinity for
the equipment surfaces and is miscible, solubilized, or
otherwise homogeneously distributed in the process
fluid, the cumulative dilution factors in Table II can be
used to estimate the concentration in the final rinse. For
example, a miscible impurity such as hydrogen peroxide,
an oxidizing cleaning agent,would be diluted by a factor
of 2 x 1016 during the six steps of the above cleaning
cycle. Thus, if the concentration of peroxide before the
pre-rinse is 10,000 ppm, its concentration in the final
rinse would be <1 part per quintillion (i.e., 1 part per
1018).
The step dilution factors for intermediate steps of the
cleaning cycle are summarized in Table III.
table III: Cumulative Dilution of the Process Fluid for Intermediate Cleaning
Steps.
The above step dilution factors for intermediate steps
can be used to simulate intermediate cleaning steps at
small scale. For example, if the pre-rinse and alkaline
wash steps are to be simulated for a product inactivation
study (16, 17), the experimental parameters should be set
to limit the dilution of the product to less than 3.8 x 105
for these steps. This would ensure that the inactivation
rate of the product at small scale for these steps repre-
sents a worst-case condition relative to that at full scale.
This is because for a given pH, temperature, and shear
Special edition: Cleaning Validation 9
Rizwan Sharnez
rate, the inactivation rate of the product is determined by
the concentration of cleaning agent and the concentra-
tion of the product, both of which depend on cumulative
dilution.
Note that the above dilutions are based on the residual
volumes for 56% glucose, a relatively viscous process
soil. This represents a worst-case condition from the
standpoint of estimating the residual volume (VR). Since
cleaning is typically performed with relatively dilute
aqueous solutions, VR for most cleaning solutions would
be substantially smaller, and therefore the dilution of the
process fluids during cleaning would be commensurate-
ly higher. Thus, the step and cumulative dilution factors
for most CIP systems are likely to be much higher than
those estimated in this example.
Conclusion
A methodology for estimating the residual volume and
dilution of the process fluid during cleaning was de-
scribed. Depending on the viscosity of the process fluid
and the angle of repose, the residual volume of a process
fluid (R) that remains on the equipment surface after
drainage was estimated to be between 4.3 and 26.5 mL/
m2. These estimates are applicable only to systems with
negligible pooling of the process fluid. Systems without
dead-legs and with surfaces sloped at an angle of at least
5 generally meet this criterion.
Based on the worst-case R value of 26.5 mL/m2 and
typical rinse and wash volumes, the cumulative and step
dilution of the process fluid during cleaning were esti-
mated to be on the order of 1016 and 105, respectively.
The cumulative dilution factor is used to estimate the
concentration of an impurity in the final rinse. The step
dilution factors are used to simulate worst-case cleaning
conditions for intermediate cleaning steps.
Methodologies for estimating fluid velocity and soil
load for small-scale cleaning characterization studies will
be described in subsequent parts of this series.
ARtICLE ACRoNYMS LIStING
CLC Cleaning characterization
d Diameter of vessel
DC Cumulative dilution of process fluid for multiple
cleaning steps
DS Step dilution of process fluid for an intermediate
cleaning step
DI Deionized
h Height up to which the process fluid wets the side
wall
R Residual process fluid per unit surface area
RD R for vessel bottom dish
RP R for miscellaneous parts of vessel
ppq Parts per quintillion (one part per 1018)
Originally published in the Spring 2011 issue of Journal of GXP Compliance
10 Special edition: Cleaning Validation
SA Surface area of equipment that comes into contact
with process fluid
SAD Surface area of bottom dish of vessel
SAP Surface area of vessel piping, filter housings, and
other miscellaneous components
SAW Surface area of vessel side wall that is wetted by
the process fluid
VR Residual volume of process fluid in equipment
VS Volume of cleaning solution
REFERENCES
1. R. Sharnez., et al, Industry Comes Clean at PDA Annual Meet-
ing, PDA Letter XLVIII (7), 28-32, July/Aug 2012.
2. B. Hoist, Developing a Cleaning Process: Cleaning in Develop-
ment, Journal of GXP Compliance 10 (3), 2006.
3. R. Sharnez, Strategies for Developing a Robust Cleaning Process
Part I: Application of Quality by Design to Cleaning, American
Pharmaceutical Review 13 (5), 77-80, 2010.
4. R. Sharnez, Validating for the Long Haul, Journal of Validation
Technology 14 (5), 2008.
5. R. Sharnez, and M. VanTrieste, Quality-by-Design for Cleaning
Validation, in Cleaning and Cleaning Validation 1, (2009) Davis
Healthcare International & PDA.
6. R. Sharnez and L. Klewer, Strategies for Developing a Robust
Cleaning Process Part II: Demonstrating Cycle Effectiveness,
American Pharmaceutical Review - Digital Edition 15 (3), 2012.
7. R. Sharnez, Taking the Guesswork out of Validation, Journal of
Validation Technology 14 (3), 2008.
8. R. Sharnez, Master Soils for Cleaning Cycle Development and
Validation: A Case Study, Cleaning and Cleaning Validation 2(2013)
Davis Healthcare & PDA.
9. A. Canhoto, A Novel Bench Scale Apparatus to Model and
Develop Biopharmaceutical Cleaning Procedures, Journal of
Validation Technology 11 (4), 2004.
10. R. Sharnez et. al, In Situ Monitoring of Soil Dissolution Dynam-
ics: A Rapid and Simple Method for Determining Worst-case Soils
for Cleaning Validation, PDA Journal of Pharm. Sc. & Tech.; 58 (4),
203-214, 2004.
11. P. Pluta, Laboratory Studies in Cleaning Validation, Journal of
Validation Technology 13 (4), 2007.
12. R. Sharnez, Leveraging Small-Scale Models to Streamline Valida-
tion, Journal of Validation Technology 14 (4), 2008.
13. R. Sharnez and L. Klewer, Parametric Release for Cleaning, Part
I: Process Characterization, Journal of Validation Technology (14) 8,
30, 2009.
14. R. Sharnez and M. Monk, Strategies for Enhancing the Perfor-
mance of Cleaning Processes Part I: A Framework for Assessing
Performance, Journal of Validation Technology 17 (1), 36-39, 2011.
15. Surface Area of Tank Heads, Mid-states Mechanical Services, Inc.,
available at: http://www.mid-statesmechanical.com/pdf/Tank%20
Heads%20Surface%20Area.pdf.
16. R. Sharnez, et al, Methodology for Assessing Product Inacti-
vation during Cleaning Part I: Experimental Approach and
Analytical Methods, Journal of Validation Technology 18 (4), 42-45,
2012.
17. R. Sharnez, J. Bussiere, D. Mytych, A. Spencer, A. To, and A.
Tholudur, Acceptance Limits for Inactivated Product based on
Gelatin as a Reference Impurity, Journal of Validation Technology
19 (1), 2013, available at: http://www.ivtnetwork.com/article/
biopharmaceutical-cleaning-validation-acceptance-limits-inacti-
vated-product-based-gelatin-re.
 Rizwan Sharnez

Experimental Parameters
for Small-Scale Cleaning
Characterization. Part II:
Effect of Fluid Velocity on
the Kinetics of Cleaning | IVT
Rizwan Sharnez, Ph.d., Angela to, S. Ravi Annapragada, Phd

ABStRACt
Methodologies for estimating experimental parameters for small-scale clean-
ing characterization are described in this series: dilution of process fluids
during clean-in-place (CIP) operations was discussed in Part I (1); the effect of
fluid velocity on the kinetics of cleaning is described in this part; the effect of
humidity, hold time and soil load on cleanability will be discussed in Part III.
The kinetics of cleaning under worst-case conditions is modeled from first
principles. The model is based on diffusion-controlled mass transfer in a
laminar falling film, which typifies worst-case cleaning conditions for CIP
operations. The effect of flowrate per unit width (Q/W) and fluid velocity
(V) on mass transfer rate in film flow is characterized. An experimental ap-
proach for optimizing Q/W and V for identifying worst-case soils for clean-
ing validation is described. The model is also used to estimate fluid velocity,
film thickness and Reynolds Number for a range of values of Q/W and angle
of inclination ().
The results indicate that Q/W and V have a relatively weak effect on the
kinetics of cleaning. For instance, when these parameters are doubled, the
mass transfer rate increases only by a factor of 8% and 12%, respectively.
The results also indicate that for 5 < < 90 and Q/W < ~1 gpm/ft (~2
mL/s/cm), the flow would be laminar and the thickness of the film would
be < ~1 mm. Further, under these worst-case conditions, V would be < ~52
cm/sec, which is substantially less than the design criterion for minimum
fluid velocity in pipes and hoses viz. 150 cm/sec (5 ft/s).

INtRodUCtIoN
Small-scale cleaning characterization data can be used to streamline valida-
tion requirements for multiproduct equipment i.e. equipment that is used
to manufacture or clean more than one product. This is accomplished by
ranking process soils associated with a given cleaning circuit based on the
relative cleanability of the soils. The hardest-to-clean or worst-case soil for
the circuit is then used to validate that circuit. This approach obviates the
need to validate the cleaning of every soil associated with a circuit. It also
facilitates the introduction of a new product into an existing multiproduct
facility. If it can be shown that the process soils associated with the new
product are easier to clean than the corresponding soils of the previously
validated product, the new product can be introduced into the facility with-
out revalidating the cleaning procedures (2).
In addition to streamlining validation requirements for multiproduct
equipment (3, 4), small-scale cleaning characterization studies can also be
used to identify suitable cleaning chemistries (5, 6), optimize cleaning pa-
rameters and processes (7, 8) and estimate cleaning times at full scale (9, 10).

Special edition: Cleaning Validation 11

Rizwan Sharnez
Experimental models for small-scale cleaning charac-
terization have been described in the literature (11-14).
In these studies, the rate at which the process residue is
removed from the surface i.e., the mass transfer rate
is measured under simulated cleaning conditions. A
critical step in the development of these models is to
identify and scale down the hardest-to-clean (worst-case)
location in the equipment (15). The worst-case loca-
tion is typically an area with poor circulation, such as a
shadowed or occluded area. Note that it is not necessary
to simulate the entire cleaning process at small scale;
instead, it is sufficient to simulate the worst-case location
within the equipment. If the process residue can be ad-
equately removed from the worst-case location, it follows
that it can also be adequately removed from other loca-
tions in the equipment. Thus, with this approach, the
cleaning times obtained at small scale would be indica-
tive of those at full scale, provided that there is adequate
spray coverage at all surfaces that need to be cleaned,
and the worst-case location is appropriately identified
and simulated at small scale.
The scalability of small-scale cleaning characteriza-
tion data depends on the accuracy with which relevant
experimental parameters are estimated. Experimental
parameters for simulating the worst-case location fall into
two distinct categories (1):
Parameters that can be readily determined from pro-
cess data such as:
Parameters that can be readily determined from
equipment and process data, such as:
o Material of construction and surface characteristics
such as roughness and curvature of coupons or
parts used to simulate large-scale equipment;
o Post-soiling parameters such as hold time, and
ambient temperature and humidity; and,
o Cleaning parameters such as rinse or wash time,
temperature of rinse solvent, and temperature and
concentration of cleaning solution.
Parameters that typically need to be determined from
first principles, such as:
o Dilution of the process fluid during cleaning (i.e.
soil to rinse solvent or cleaning solution ratio).
o Velocity of rinse solvent or cleaning solution at the
worst-case location the subject of this paper.
o Soil load.
Flowrate and Fluid Velocity at Worst-Case Location
Engineering standards provide guidelines for setting the
minimum average velocity or minimum volumetric flow
rate of rinse solvent or cleaning solution in various sec-
tions of a CIP circuit. For instance, it is recommended
that for pipes and hoses the minimum fluid velocity
VMIN be 5 ft/s (1.5 m/s), and for film flows in vessels
the minimum flow rate per unit width (Q/W)MIN be
2.5 gpm per ft of vessel circumference (31 L/min/m) (16).
12 Special edition: Cleaning Validation
These criteria are designed to ensure turbulent flow dur-
ing CIP; the 5 ft/s criterion for VMIN is also designed to
ensure flooding in pipes and hoses.
For pipes and hoses, the 5 ft/s criterion for VMIN can
be readily met at all locations and is therefore relatively
straightforward to simulate at small scale. For film flows,
however, the flow rate per unit width at the worst-case
location (Q/W)WCL such as the underside of an impel-
ler blade or a magnetically coupled bottom-mounted
impeller is likely to be substantially less than (Q/W)
MIN. Design and operational variables that contribute to
(Q/W)WCL being less than (Q/W)MIN are summarized in
Table 1.
table I: Design and operational variables that contribute to the flow rate
per unit width at the worst-case location (Q/W)WCL being substantially less
than the recommended minimum operating value (Q/W)MIN. For vessels, the
recommended value for (Q/W)MIN is 2.5 gpm per foot of
vessel circumference.
Effect of Flowrate and Fluid Velocity on Kinetics and
Cleaning
The mass transfer rate from a stationary surface into a
laminar falling film of a Newtonian fluid was investi-
gated by Kramers and Kreyger for a flat rectangular layer
(17, 18) and by Blount for viscous drops (19, 20). Their
results indicate that the mass or molar flux (NAX) of the
solute (A) into the fluid (X) is a function of the solubil-
ity (SAX) and diffusivity (DAX) of the solute in the fluid;
the density (), dynamic viscosity () and flowrate per
unit width (Q/W) of the fluid; and the length (L) of the
surface along the direction of the flow (Figure 1):
NAX = k SAX (DAX L)2/3 (Q/W)1/9 ()-2/9 Equation [1a]
where is /, the kinematic viscosity; and k is a con-
stant that includes the acceleration due to gravity (g), the
angle of inclination (), and the width (W).
Figure 1: Mass transfer in gravity-driven film flow: Solute A diffusing
into a laminar falling film of fluid X, moving with a fully developed para-
bolic velocity profile.

For a surface of given length, and a given fluid viscosity
and angle of inclination,
NAX (SAX DAX2/3) (Q/W)1/9 Equation [1b]
Further, for laminar falling films,
Q/W V3/2
where V is the average fluid velocity (21); thus,
NAX (SAX DAX2/3) V1/6 Equation [1c]
The flux (NAX) of the solute into the fluid is the rate at
which the solute is removed from the surface. Thus, NAX
is effectively a measure of the kinetics of cleaning. Note
that the effect of Q/W and V on the mass transfer rate
and therefore the kinetics of cleaning is relatively weak.
For example, when Q/W or V is doubled, the mass
transfer rate increases only by a factor of 21/9 (8%), and
21/6 (12%), respectively.
Equation 1 is valid when (1) the Reynolds Number Re
= 4(Q/W)/ < 1500, the criterion for laminar flow in a
falling film; (2) the velocity profile in the falling film is
fully developed, a condition that holds when L >> , the
thickness of the film; and (3) the distance over which the
solute diffuses into the film (d) is << , and as a result,
the velocity profile for 0 < y < d can be approximated
as a linear function of distance from the surface being
cleaned (y) (Figure 1). In terms of the parameters in
Equation 1, the third condition is satisfied when the
solubility (SAX) and/or diffusivity (DAX) of the solute in
the fluid are low enough for the mass transfer to be diffu-
sion controlled.
The above conditions would be satisfied at the
worst-case location in the equipment for a process soil
that is difficult to clean, as this represents a worst-case
scenario from the standpoint of cleaning viz. diffusion-
controlled mass transfer in a laminar falling film. Thus,
Equation 1 can be used to characterize the effect of
process parameters such as Q/W and V on the kinetics
of cleaning under worst-case conditions and for design
purposes. It should also be noted that since Equation
1 is derived for mass transfer of a single component (A)
into a pure solvent (X), its applicability to complex multi-
component process soils or solvents containing formulat-
ed cleaning agents would require the use of an effective
solubility and diffusivity in the cleaning solution. Con-
sequently, Equation 1 cannot be readily used to predict
the cleanability of multicomponent soils; nonetheless, it
can be used to characterize the effect of process param-
eters on the kinetics of cleaning, and to thereby identify
and establish meaningful operating ranges for critical
process parameters. Further, this equation can also be
used to develop experimental models for cleaning (2).
Rizwan Sharnez
An experimental approach for optimizing flowrate
and fluid velocity for evaluating relative cleanability at
small scale is described in the next section.
Flow Rate and Fluid Velocity for Evaluating Relative
Cleanability
Consider a system that is validated to manufacture and
clean product A. A new product (B) needs to be manu-
factured and cleaned in the same equipment. A small-
scale study is performed to evaluate the cleanability of
A relative to that of B. If A is harder to clean than B, the
new product could be introduced without revalidation.
The objective is to determine the optimum flowrate per
unit width (Q/W) and fluid velocity (V) for the small-
scale study.
For products A and B, Equation 1b can be written as
follows:
NAX (SAX DAX2/3) (Q/W)1/9 Equation [2a]
NBX (SBX DBX2/3) (Q/W)1/9 Equation [2b]
Thus,
NAX / NBX = (SAX DAX2/3) / (SBX DBX2/3) Equation [2c]
Since the cleaning time (t) is inversely proportional to
the mass transfer rate (N), the cleanability of B (tB) rela-
tive to that of A (tA) can be expressed as
tB / tA = NAX / NBX = (SAX DAX2/3) / (SBX DBX2/3) Eq [2d]
Equation 2d indicates that relative cleanability (tB/
tA) depends on the physical properties of A and B (viz.
the solubility and diffusivity of A and B in the fluid).
Further, relative cleanability is independent of Q/W, and
therefore of V. Thus, if the objective of the study is to
rank A and B based on cleanability, Q/W and V can be
set to any reasonable value, provided that the resulting
flow is laminar in this case Re = 4(Q/W)/ < ~1000.
In practice, however, a lower value of Q/W is preferable
because the absolute difference between tA and tB (tAB)
is amplified, which in turn makes it commensurately
easier to differentiate between the two soils based on the
larger magnitude of tAB. If necessary, tAB can be am-
plified by reducing Q/W up to the point where the film
is still intact and uniform, i.e. it does not disintegrate into
slower-moving unsteady drops.
An equation for estimating fluid velocity of a laminar
falling film from Q/W and is derived in the next sec-
tion.
Estimation of Fluid Velocity in a Laminar Falling Film
A laminar falling film of a Newtonian fluid flowing pri-
marily under the influence of gravity is shown in Figure
1. The flow is delineated as a thin sheet of liquid flowing
down an inclined flat plate of length L and width W. As
Special edition: Cleaning Validation 13
Rizwan Sharnez
the liquid flows down the plate, it forms a film of thick-
ness and develops a parabolic velocity profile, with the
maximum velocity at the film surface. For this type of
flow, the average fluid velocity (V) and film thickness ()
can be expressed as follows (21):
Equation 3
Equation 4
Where is the density of the liquid, g is acceleration due
to gravity, is the angle of, is the dynamic viscosity of
the fluid, is the mass flow rate, and W is the width of
the film.
Equations 3 and 4 can be combined to eliminate and
express V in terms of measurable parameters:
Equation 5
Where Q is /, the volumetric flow rate, and is /,
the kinematic viscosity of the fluid.
Equation 5 is valid under the following conditions: (1)
when edge effects are negligible, a condition that is valid
when L and W are >> ; and (2) when viscous forces are
large enough to prevent continued acceleration of the
liquid along the length of the plate i.e., at a low Reyn-
olds Number, when the flow is laminar. Under these
conditions, V is independent of the distance traversed
along the incline (L). Note that at the worst-case location
the above conditions would be satisfied because (a) the
surfaces being cleaned are relatively large, and thus L and
W would be >> ; and (b) the flow would be laminar.
The Reynolds number (Re) is used to classify a fall-
ing film into three flow regimes: (a) laminar flow with
negligible rippling (Re < 20); (b) laminar flow with
pronounced rippling (20 < Re < 1500); and (c) turbulent
flow (Re > 1500). When Re is less than 20, the ripples
are very long and grow slowly down the surface of
the film. As Re increases above 20, the ripple growth
increases rapidly. Because of the assumptions made
in developing the above model (21), the error in using
Equation 5 to estimate velocity increases with ripple
growth and Re. The velocities estimated using Equa-
tion 5 have been shown to be in good agreement with
experimentally observed velocities when Re is less than
1000 (22, 23).
The average velocity of the cleaning solution is esti-
mated from the flowrate per unit width (Q/W) and the
angle of inclination () using Equation 5. The estimates
are based on the kinematic viscosity () of water at 20C
(0.01 cm2/s), acceleration due to gravity (g) of 981 cm/
s2, and a range of values of Q/W and . The results,
summarized in Table 2, indicate that for 5 < < 90
and Q/W < ~1 gpm/ft (~2 mL/s/cm), the flow would be
laminar and the thickness of the film ( would be < ~1
14 Special edition: Cleaning Validation
mm, if the film were stable (i.e. if it did not disintegrate
into unsteady drops). Further, under these conditions,
the average velocity of the fluid would be < ~52 cm/sec,
which is substantially less than the design criterion for
VMIN in pipes and hoses viz. 150 cm/sec (5 ft/s).
table II: Average velocity, film thickness, Reynolds Number and relative
mass transfer rate in a laminar falling film for a range of values of flowrate
and angle of inclination.
CoNCLUSIoN
Small-scale experimental models are used to determine
worst-case soils for cleaning validation, and estimate
cleaning times and other performance parameters. A
critical step in the development of these models is to
identify and scale down the hardest-to-clean or worst-
case location in the equipment. For cleaning operations,
the worst-case location is typically an area within the
equipment with poor circulation, such as a shadowed or
occluded area. Examples of such locations include the
underside of a probe or an impeller blade where there
is no direct or indirect impingement of the cleaning
solution during CIP operations. Instead, the flow of the
fluid at the worst-case location is in the form of a laminar
falling film.
A mathematical model for diffusion-controlled mass
transfer in a laminar falling film was used to character-
ize the effect of flowrate per unit width (Q/W) and fluid
velocity (V) on the kinetics of cleaning under worst-case
conditions. The results indicate that the effect of these
parameters on the rate of mass transfer and therefore
the kinetics of cleaning is relatively weak. The mass
transfer rate increases only by a factor of 8% and 12%,
respectively, when Q/W or V is doubled.
An experimental approach for optimizing Q/W and
V for evaluating relative cleanability at small scale was
described. Relative cleanability was shown to depend on
the solubility and diffusivity of the soils being compared.
Further, for diffusion-controlled mass transfer which
typifies worst-case cleaning conditions for CIP opera-

tions relative cleanability was found to be independent
of Q/W and V. Thus, if the objective of the study is to
rank coils based on cleanability, Q/W and V can be set
to any reasonable value, provided that the resulting flow
is laminar. In practice, however, a lower value of Q/W
is preferable because the absolute difference between the
cleaning times of the soils (tAB) is amplified, and as a
result, the ability to differentiate between the soils based
on the larger magnitude of tAB is enhanced commen-
surately. If necessary, tAB can be amplified by reducing
Q/W up to the point where the film is still intact and
uniform i.e. it does not disintegrate into slower-moving
unsteady drops.
The laminar falling film model was also used to esti-
mate fluid velocity (V), film thickness () and Reynolds
Number (Re) from Q/W and the angle of inclination
(). The results indicate that for 5 < < 90 and Q/W
< ~1 gpm/ft (~2 mL/s/cm), the flow would be laminar
and would be < ~1 mm, if the flow was stable. Further,
under these conditions, V would be < ~52 cm/sec, which
is substantially less than the design criterion for VMIN in
pipes and hoses viz. 150 cm/sec (5 ft/s). The calculated
values of V have been shown to be in good agreement
with experimentally observed velocities when Re is less
than 1000.
SYMBoLS ANd ACRoNYMS
CIP Clean-in-place
D Diffusivity
g Acceleration due to gravity
L Length of object being cleaned
N Mass or molar flux
Q Volumetric flow rate
Re Reynolds number
S Solubility
t Time
V Average velocity of cleaning solution
W Width of laminar falling film
Angle of inclination (slope)
Film thickness
Dynamic Viscosity
Kinematic viscosity
Density
Mass flow rate
SUBSCRIPtS
A Product A
B Product B
MIN Minimum
WCL Worst-case location
X Fluid X
Rizwan Sharnez
REFERENCES
1. R. Sharnez, A. To, and A. Tholudur, Experimental Parameters for
Small-scale Cleaning Characterization Part I: Dilution of Process
Fluids during Cleaning, Journal of Validation Technology 19 (3),
2013.
2. R. Sharnez, Leveraging Small-Scale Models to Streamline Valida-
tion, Journal of Validation Technology 14 (4), 2008.
3. R. Sharnez, Taking the Guesswork out of Validation, Journal of
Validation Technology 14 (3), 2008.
4. R. Sharnez, Master Soils for Cleaning Cycle Development and
Validation: A Case Study, Cleaning and Cleaning Validation 2(2013)
Davis Healthcare & PDA.
5. R. Sharnez, et al., Industry Comes Clean at PDA Annual Meet-
ing, PDA Letter XLVIII (7), 28-32, July/Aug 2012.
6. B. Hoist, Developing a Cleaning Process: Cleaning in Develop-
ment, Journal of GXP Compliance 10 (3), 2006.
7. R. Sharnez, Strategies for Developing a Robust Cleaning Process
Part I: Application of Quality by Design to Cleaning, American
Pharmaceutical Review 13 (5), 77-80, 2010.
8. R. Sharnez, Validating for the Long Haul, Journal of Validation
Technology 14 (5), 2008.
9. R. Sharnez and M. VanTrieste, Quality-by-Design for Cleaning
Validation, in Cleaning and Cleaning Validation 1, (2009) Davis
Healthcare International & PDA.
10. R. Sharnez and L. Klewer, Strategies for Developing a Robust
Cleaning Process Part II: Demonstrating Cycle Effectiveness,
American Pharmaceutical Review - Digital Edition 15 (3), 2012.
11. Canhoto, A Novel Bench Scale Apparatus to Model and Develop
Biopharmaceutical Cleaning Procedures, Journal of Validation
Technology 11 (4), 2004.
12. R. Sharnez et al, In Situ Monitoring of Soil Dissolution Dynam-
ics: A Rapid and Simple Method for Determining Worst-case Soils
for Cleaning Validation, PDA Journal of Pharm. Sc. & Tech. 58 (4),
203-214, 2004.
13. P. Pluta, Laboratory Studies in Cleaning Validation, Journal of
Validation Technology 13 (4), 2007.
14. R. Sharnez and L. Klewer, Parametric Release for Cleaning, Part
I: Process Characterization, Journal of Validation Technology (14) 8,
30, 2009.
15. R. Sharnez and M. Monk, Strategies for Enhancing the Perfor-
mance of Cleaning Processes Part I: A Framework for Assessing
Performance, Journal of Validation Technology 17 (1), 36-39, 2011.
16. ASME Bioprocessing Equipment (BPE) Standard, 2012.
17. Kramers, H., and P. J. Kreyger, Mass Transfer between a Flat
Surface and a Falling Film, Chemical Engineering Science, Vol. 6,
pp. 42-48 (1956).
18. R. Bird, W. Stewart, and E. Lightfoot, Transport Phenomena, 2nd
Ed., 2007. p. 562-563.
19. M. Blount, Aspects of advection-diffusion-reaction flows of
relevance to decontamination, KTN Internship Report, (2010).
20. J. Landel, H. McEvoy, and S. Dalziel, Cleaning of Viscous Drops
on a Flat Inclined Surface Using Gravity-Driven Film Flows,
Food and Bioproducts Processing, Vol 93, p. 310-317 (2015).
21. R. Bird, W. Stewart, and E. Lightfoot, Transport Phenomena. 2nd
Ed., 2007. pg.46.
22. West and Cole, Surface velocities of thin liquid films, Chemical
Engineering Science, 22, 1388-1389, 1967.
23. S. Portalski, Velocities in film flow of liquids on vertical plates,
Chemical Engineering Science 19, 575-582, 1964.
Special edition: Cleaning Validation 15
Rizwan Sharnez

Methodology for Assessing

Product Inactivation during
Cleaning Part I: Experimental
Approach and Analytical
Methods | IVT
Rizwan Sharnez, Ph.D

ABSTRACT
For multiproduct cleaning validation, the conventional approach for setting
an acceptance limit for the process residue is based on the maximum allow-
able carryover (MAC) of the active pharmaceutical ingredient (API) (de-
pending on the process soil, API refers to the active pharmaceutical ingredi-
ent in the drug product, drug substance, or drug substance intermediate).
However, if the API becomes pharmacologically inactive during cleaning
the acceptance limit does not need to be based on active product. This is
an important consideration in biopharmaceutical manufacturing because
the cleaning conditions are generally aggressive enough to inactivate the
product.
The experimental approach and analytical methods for assessing
inactivation of the API during cleaning are described in Part I. A rational
approach for setting safety-based acceptance limits for inactivated product
and process residuals is described in Part II. The scope of this paper is
limited to biopharmaceutical cleaning processes; nonetheless, the under-
lying concepts may be useful in designing inactivation studies and setting
acceptance limits for other types of pharmaceutical cleaning processes.

INTRODUCTION
An important regulatory expectation for multiproduct cleaning validation is
to demonstrate that potential carryover of the previously manufactured API
(Product A) into the subsequently manufactured product (Product B) is be-
low an acceptable level. This criterion is often assessed through a maximum
allowable carryover (MAC) calculation for the previously manufactured API
(1-5). The MAC calculation is typically based either on the minimum thera-
peutic dose (1), or the acceptable daily exposure (ADE) (2) of the previously
manufactured API.

Limitations of the MAC Approach

A limitation of the conventional MAC approach is that it is based on the
assumption that the product is active after the cleaning. This has important
implications for biopharmaceutical manufacturing because the API is often
inactivated by the cleaning process (6, 7).
Another limitation of the MAC approach is that the calculated accep-
tance limits are often below the limit of quantitation (LOQ) of non-
specific analytical methods, such as total organic carbon (TOC). The
LOQ of TOC- based methods is typically between 0.05 and 0.2 ppm. The
large surface areas and small batch sizes involved in biopharmaceutical
manufacturing further exacerbate this issue. Product specific immunoas-

16 Special edition: Cleaning Validation


says (PSIA) such as ELISA and EIA have been used
to address this issue; the LOQ of most PSAs is on
the order of 10 ppb. PSIAs detect activity indirectly
by recognizing specific epitopes (short sequences of
amino acids ) in the API; however, epitopes are known
to degrade during cleaning, and thus the results can be
misleading (8, 9).
Other limitations of the MAC approach are dis-
cussed in the literature (9).
PRODUCT INACTIVATION APPROACH
With the product inactivation approach, if the API is in-
activated during cleaning, the acceptance limits may be
set based on the inactivated product instead of the API.
The product inactivation approach is therefore more re-
flective of the phenomenological aspects of the cleaning
process. Additionally, the acceptance limits based on in-
activated product are very unlikely to be below the LOQ
of TOC (refer to Part II). Thus, the product inactivation
approach alleviates the limitations of the MAC approach
described in the previous section.
The methodology described in Part I includes
experimental simulation of the cleaning processes at
small scale and analytical methods to evaluate inacti-
vation of the API during cleaning. A rational approach
for setting safety-based acceptance limits is described
in Part II.
PROPOSED METHODOLOGY
Inactivation of the product during cleaning has impor-
tant implications for cleaning validation of multiproduct
equipment. If it can be demonstrated that the product
becomes pharmacologically inactive during cleaning,
there is limited value in verifying removal of the active
ingredient. Instead, it is more appropriate to demonstrate
that the inactivated product has been removed below a
predefined acceptance limit. This is consistent with the
expectation that the carryover of an extrinsic impurity
into a subsequent batch should be justified from the
standpoint of the safety and efficacy of the product. It
also obviates the need to develop PSIAs for cleaning
validation.
Biopharmaceutical cleaning cycles are generally
designed to expose product contact equipment to
extremes of pH (<2 and >13) and temperature (60-
80C) for several minutes. Under these conditions
monoclonal antibodies, therapeutic proteins, and
other biopharmaceuticals are known to degrade and
denature rapidly, and are therefore likely to become
pharmacologically inactive (6, 7). The product inac-
tivation approach should therefore be considered for
biopharmaceutical cleaning validation.
GUIDANCE FOR DESIGNING INACTIVATION STUDIES
Fragmentation and inactivation of an API during clean-
ing can be assessed by exposing the process soil to
Rizwan Sharnez
worst-case cleaning conditions at bench scale (10, 11).
The results of the bench-scale studies can justifiably be
extrapolated to the full-scale cleaning process. That is
because under worst-case cleaning conditions of laminar
flow and low shear rate fragmentation and inactivation
are independent of scale (i.e., they depend on cleaning
parameters that are not a function of the of spatial coor-
dinates of the system, such as time, temperature, concen-
tration, and the ratio of cleaning solution to process soil).
The bench-scale experiments are typically per-
formed in a vial or dialysis cassette, and are designed
to simulate full-scale cleaning conditions that are least
conducive (worst-case) for inactivation. For example,
for a chemical wash, the lowest applicable concentra-
tion of cleaning agent, temperature, duration, and ratio
of cleaning solution to residual process soil should be
considered in simulating the cleaning cycle at bench
scale. Other operating parameters that can contribute
to product inactivation include dirty hold time (DHT)
and associated drying conditions (humidity and air
circulation rate), and shear rate due to impingement
and turbulence.
An operating parameter or step can be eliminated
from the experimental design if its elimination repre-
sents a worst-case scenario from the standpoint of in-
activation. This approach can be leveraged to simplify
the bench-scale studies. For instance, if it is reasonable
to assume that product inactivation increases with
shear rate, then it can be eliminated from the experi-
mental design (i.e., the shear rate need not be simu-
lated in the experiment). Similarly, the ratio of cleaning
solution to process soil can be reduced, and the acid
wash and rinse steps can be eliminated to minimize
dilution of the process soil, and facilitate detection of
the process residue in the sample. When making such
changes, unexpected effects such as aggregation of the
API can occur. It is therefore important to make sure
that the modifications do not result in experimental
artifacts.
If the cleaning cycles are being developed or modi-
fied, the inactivation study should be designed to
evaluate the effect of key operating parameters on the
fragmentation and inactivation rate of the API. This in-
formation, together with data from cleanability studies
(12, 13), can be used to identify cycle parameters that
are effective in inactivating the API.
For existing cleaning cycles, the cleaning conditions
for the inactivation study should be based on worst-
case operating parameters for all systems involved. For
instance, if different systems are cleaned with differ-
ent cleaning solutions and at different temperatures,
then the study should be performed with the mildest
cleaning solution, at the lowest cleaning agent concen-
tration, and the lowest temperature, if these conditions
are least conducive for inactivation. Further, for clean-
in-place (CIP) systems with multiple toggle paths, the
Special edition: Cleaning Validation 17
Rizwan Sharnez
duration of cleaning should be based on the toggle
path with the shortest cleaning time.
After exposing the process soil to worst-case clean-
ing conditions, the samples are titrated to a neutral
pH, and cooled to 4C to minimize further fragmen-
tation and inactivation of the API. The samples are
then subjected to analytical testing as described in the
next section. If the API is inactivated when exposed to
worst-case cleaning conditions, then the acceptance
limit for the inactivated product can be set based on
the approach described in Part II.
If the results indicate that the API is not inactivated
during cleaning, then the acceptance limits should be
set based on the acceptable carryover of the API (1-2).
If the API is partially inactivated, then the acceptance
limits should be determined for the API, as well as for
the inactivated product, and the lower of the two limits
should be used. Alternatively, the cleaning parameters
can be modified to ensure inactivation of the API. This
can be facilitated by running additional studies to
characterize the effect of specific cleaning parameters
on the API.
RECOMMENDED ANALYTICAL METHODS
Analytical methods commonly used to evaluate the effect
of cleaning parameters on the previously manufactured
API are described in this section. These methods are
used to evaluate fragmentation and inactivation of the
API at bench scale, and to detect target impurities (in this
case, the previously manufactured API and/or inactivated
product in the process residue) in cleaning validation
samples. The analytical results are used to understand
the impact of the cleaning conditions on the process soil,
and to set rational safety-based acceptance limits for the
target impurities (Part II). The detection methods are
used to verify that the concentrations of target impurities
in cleaning validation samples are below their respective
acceptance limits.
Sodium dodecyl sulphate polyacrylamide gel elec-
trophoresis (SDS-PAGE) or capillary electrophoresis
(CE) are generally used to characterize fragmentation
of the API during cleaning. For SDS-PAGE, a 4 to 20
percent gradient corresponds to a molecular weight
range of 4 to 250 kDa, which is sufficient for most
biological APIs. While CE provides greater sensitivity,
lower variability due to the absence of staining, and
high throughput capability as compared to SDS-PAGE,
both methods are adequate for demonstrating distinct,
size-based separation of fragmented protein. Size
exclusion high-pressure liquid chromatography (SE-
HPLC) can also be utilized for size-based separation of
protein fragments; however, it can be difficult to obtain
a distinct size-based separation across a wide range of
fragment sizes.
Inactivation of the API can be evaluated by meth-
ods that measure loss of biological activity or function
18 Special edition: Cleaning Validation
(binding sites that are functionally intact), such as a
bioassay. These methods measure the relative amount
of biologically active product. Thus, they can be used
to evaluate the degree of inactivation of the API during
cleaning.
A non-specific method such as total organic carbon
(TOC) can be used to detect target impurities in clean-
ing validation samples. TOC has an LOQ of approxi-
mately 0.2 ppm, which is sufficient for most cleaning
applications. The use of a non-specific method allows
for the detection of both intact API and inactivated
product.
With the above methods, appropriate standards and
untreated controls should be included to provide a
basis for comparison, and to assess the impact of any
experimental artifacts and potential matrix effects.
For SDS-PAGE, appropriate MW markers should be
included to facilitate comparison of the fragments to
the untreated controls.
CONCLUSION
An important consideration in multiproduct cleaning
validation is to demonstrate that the carryover of the
previously manufactured API into a batch of the subse-
quently manufactured product is below an acceptable
limit. This criterion is often met through a MAC assess-
ment for the API; however, if the previously manufac-
tured API becomes pharmacologically inactive during
cleaning, the acceptance limit does not need to be based
on active product. This is an important consideration in
biopharmaceutical manufacturing because the cleaning
conditions are generally aggressive enough to inactivate
the product.
Fragmentation and inactivation of an API during
cleaning can be assessed by exposing the process
soil to simulated cleaning conditions at bench scale
(10, 11). The bench scale experiments are typically
designed to simulate full-scale cleaning conditions that
are least conducive (worst case) for inactivation. The
degree of inactivation is evaluated by subjecting the
sample and untreated controls to the appropriate as-
says, (e.g., SDS-PAGE and bioassay are used to evaluate
fragmentation and biological activity, respectively). The
results of the study are used to set appropriate accep-
tance limits for cleaning validation.
If the API is inactivated during cleaning, the accep-
tance limits for the process residuals can be set based
on the approach described in Part II.
ACKNOWLEDGEMENTS
We thank Arun Tholudur, Aine Hanley, and Sam Guhan
of Amgen; Rich Kemmer and Paul Whetstone of Bayer;
James Crawford and Michael Maurer of GlaxoSmith-
Kline; John Krayer of Janssen; Josh Getchell of Lonza;
David Barabani and Michael Parks of Pfizer; and Markus
Bluemel of Novartis for their help and support.

REFERENCES
1. G.L. Fourman and M.V. Mullen, Determining Cleaning Valida-
tion Acceptance Limits for Pharmaceutical Manufacturing
Operations, Pharmaceutical Technology 17 (4), 54-60, 1983.
2. ISPE, Risk-Based Manufacture of Pharmaceutical Products:
A Guide to Managing Risks Associated with Cross-Contami-
nation 7, International Society for Pharmaceutical Engineers
(ISPE). Tampa, FL, First ed., 2010.
3. R. Sharnez, Strategies for Setting Rational MAC-based Limits
Part I: Reassessing the Carryover Criterion, Journal of Valida-
tion Technology 16 (1), 71-74, 2010.
4. R. Sharnez, A. To, and L. Klewer, Strategies for Setting Ratio-
nal MAC-based Limits Part II: Application to Rinse Samples,
Journal of Validation Technology 17 (2), 43-46, 2011.
5. R. Sharnez, and A. To, Strategies for Setting Rational MAC-
based Limits Part III: Leveraging Toxicology and Cleanability
Data, Journal of Validation Technology 17 (3), 24-28, 2011.
6. R. Sharnez, and A. To, Cleaning Validation of Multiproduct
Equipment: Acceptance Limits for Inactivated Product, Part
I The Comparable Quality Approach, Journal of Validation
Technology 17 (4), 32-36, 2011.
7. R. Sharnez, E. Aisenbrey, J. Bercu, D. Binkley, and A. Tholudur,
Cleaning Validation of Multiproduct Equipment: Acceptance
Limits for Inactivated Product, Part II- Application of the Com-
Rizwan Sharnez
parable Quality Approach to Intrasite Assessments, Journal of
Validation Technology 18 (2), 17-25, 2012.
8. Health Canada, Cleaning Validation Guidelines (GUIDE-0028);
Section 8.3, Health Products and Food Branch Inspectorate,
2008, available at: http://www.hc-sc.gc.ca/dhp-mps/compli-
conform/gmp-bpf/validation/index-e....
9. R. Sharnez, M. Horner, A. Spencer, and A. Tholudur, Leverag-
ing Acceptable Exposure of Host Cell Protein to Set Acceptance
Limits for Inactivated Product, Journal of Validation Technology
18 (3), 38-44, 2012.
10. K. Kendrick, A. Canhoto, and M. Kreuze, Analysis of Degrada-
tion Properties of Biopharmaceutical Active Ingredients as
Caused by Various Process Cleaning Agents and Temperature,
Journal of Validation Technology 15 (3), 69, 2009.
11. N. Rathore, W. Qi, C. Chen, and W. Ji, Bench-scale character-
ization of cleaning process design space for biopharmaceuti-
cals, Biopharm International 22 (3), 2009.
12. R. Sharnez, Dont Bet on Quality-by-Chance: Part II Lever-
aging Small-Scale Models to Streamline Validation, Journal of
Validation Technology 14 (4), 2008.
13. R. Sharnez and L. Klewer, Strategies for Developing a Robust
Cleaning Process Part II: Demonstrating Cycle Effectiveness,
American Pharmaceutical Review - Digital Edition 15 (3), 2012.
JVT
Special edition: Cleaning Validation 19
Rizwan Sharnez

Methodology for Assessing

Product Inactivation During
Cleaning Part II: Setting
Acceptance Limits of
Biopharmaceutical Product
Carryover for Equipment
Cleaning | IVT
By Adam Mott, Bill Henry, Edward Wyman, Greg Randall, Kathleen Bellorado, Markus Blmel, Mary
Ellen Clark, Michael Parks, Ronan Hayes, Scott Runkle, Wendy Luo

ABSTRACT
For multi-product biopharmaceutical facilities, setting the acceptable level of
process residues following equipment cleaning is an important regulatory,
business, product quality, and patient safety consideration. Conventional
approaches for setting an acceptance limit for process residues have been
based on the assumption that the active pharmaceutical ingredient (API)
(depending on the process soil, API refers to the active pharmaceutical in-
gredient in the drug product, drug substance, or drug substance intermedi-
ate) is chemically or functionally intact following the cleaning process. These
approaches include Maximum Allowable Carryover (MAC) Health Based
Exposure Limits and other dose or Permissible Daily Exposure (PDE)-
based limits. The concept for cleaning acceptance limits based on intact
product originated from the manufacturing of small molecule pharmaceu-
ticals (1). In contrast to pharmaceutical small molecules, biopharmaceutical
products are large molecules that are likely to degrade and become inactive
when exposed to cleaning conditions. Therefore, an alternative approach to
setting cleaning acceptance limits for biopharmaceutical products based on
the actual process residues that could potentially be present on production
equipment should be considered.
Part I described the methodology to assess and verify API inactivation
during cleaning (2). In Part II, alternative approaches for setting accept-
able levels of process residue will be described building upon the basis
that API inactivation by the cleaning process has been demonstrated.

INTRODUCTION
When multiple products are manufactured using the same equipment, it is
important to ensure that potential product or process residues from the pre-
viously manufactured batch are removed to an acceptable level to ensure the
subsequently manufactured product will not be impacted. The acceptable
level of carryover has often been based on the active, intact API. However,
for biopharmaceutical products, the API typically degrades and becomes
pharmacologically inactive during cleaning, and therefore the cleaning ac-
ceptance criteria do not need to be based on the concept of intact and active
product. Rather, the cleaning acceptance limit should be based on potential
process residues that have a greater carryover potential founded on phe-
nomenological aspects of the cleaning process. The scope of this paper tar-

20 Special edition: Cleaning Validation


gets biopharmaceutical APIs; nonetheless, the underlying
concepts may be useful in setting acceptance limits for
other types of pharmaceutical products where inactiva-
tion during the cleaning process can be demonstrated.
This paper will include a review of product inacti-
vation, information on product detection using total
organic carbon (TOC), and alternative approaches
for setting acceptance limits for equipment cleaning.
The intention of this paper is to propose acceptable
approaches for setting cleaning limits for biopharma-
ceutical process equipment that may be considered.
However, it should not be considered prescriptive for
what approach is most appropriate or should be used
since every production facility, processes, and products
manufactured are unique.
PRODUCT INACTIVATION
Biopharmaceuticals are large molecule drug products
(e.g., monoclonal antibodies, therapeutic proteins, etc.)
that are made in processes using living organisms rather
than extracted from a native source or by synthesizing
compounds. The equipment cleaning cycles are designed
to expose product contact areas to cleaning detergents
that include alkaline and acidic chemicals. Under these
exposure conditions, the high pH in alkaline chemi-
cals (typically pH >11) and low pH in acidic chemicals
(typically pH <2) are efficient in hydrolyzing biological
peptide bonds, rendering biopharmaceutical products
biologically inactive by degradation and denaturation.
If it is demonstrated that the product becomes pharma-
cologically inactive during cleaning, there is no longer a
risk of active product carryover and, furthermore, a lim-
ited value in verification of the removal of active product
from equipment surfaces.
It should be noted that an antibody-drug-conjugate
(ADC) is considered a biopharmaceutical product,
but it contains an extremely toxic small molecule that
attaches to a protein through organic linkers. Due to
the functional and toxicological behavior of an ADC
product, specifically the toxic small molecules attached
to the large molecule, PDE limits should be established
for ADC products based on the toxicity of the conju-
gate; therefore, they are not in the scope of this paper.
Part I discussed experimental approaches and ana-
lytical methods that can be used to evaluate product
inactivation by the cleaning detergents. This important
first step characterizes the biological activity of the API
and may also be used to gain a further understanding
of remaining product fragments.
Inactivated Product Rinsibility/Removal
The inactivated product and/or product fragments may
be further evaluated to better understand the effect of
the cleaning process and the potential for carryover. The
final step in most, if not all, cleaning procedures is a
Rizwan Sharnez
final rinse of higher grade water quality, typically Water
for Injection (WFI). The volume and flow rate of this
rinse are designed to be sufficient to remove all residual
cleaning agent(s) to a conductivity level approaching the
WFI source water. The inactivated product that results
from exposure to the cleaning conditions is likely to be
more water-soluble than the intact protein due to its de-
creased size (3) and, therefore, should be readily rinsed
from equipment surfaces in the last step of the cleaning
process. The rinsibility or ease of removal of inacti-
vated product/product fragments may be evaluated in a
rinsibility study, where the inactivated product material
is spiked onto representative coupons and exposed to a
worst-case (e.g., no impingement, lower flow rate, etc.)
water rinse in comparison to full scale cleaning cycles.
If the worst-case rinse removes the product spike from
the coupon, it demonstrates that the inactivated product
fragments are not a carryover concern.
The Product Inactivation Study demonstrates the
product is not active after exposure to cleaning condi-
tions. The rinsibility study demonstrates that the
potential product fragments created from exposure of
the product to cleaning conditions are not a carryover
risk. Therefore, setting acceptance limits for equip-
ment cleanliness based upon intact product activity or
potential product fragments would not be reflective of
the actual residuals that are most likely to be present
on equipment after cleaning based upon the phenom-
enological effects of the cleaning process.
DETECTION OF PRODUCT OR PROCESS RESIDUES
Most biopharmaceutical process components (e.g.,
API, host cell proteins, media, and cleaning detergents)
include organic carbon within their composition. The ap-
plication of TOC as the post-cleaning detection method
for product carryover is considered more stringent than
a product-specific method as it would detect all process/
cleaning residuals containing carbon, including poten-
tially difficult to remove materials. The TOC analysis
method is relatively sensitive (scale of ppb limits of detec-
tion and quantitation) that can be used for swab samples,
rinse samples, and inline monitoring.
The approaches included in this paper for assessing
equipment cleanliness are based on TOC, but they can
be adapted to product specific methods if required.
SETTING THE ACCEPTANCE LIMITS
Four different approaches for setting cleaning acceptance
limits will be discussed. Each limit setting approach
(Cleaning Process Capability, Safety Factor, Toxicology
Threshold, and Performance Control) ensures patient
safety and no impact to subsequent product quality. The
assumption inherent in each approach is that prod-
uct inactivation from the cleaning process conditions
has been demonstrated, which provides the scientific
rationale and assurance of no active product carryover.
Special edition: Cleaning Validation 21
Rizwan Sharnez

Every facility has unique characteristics, products, and
operational variables to consider. The following ap-
proaches are not intended to be inclusive of all acceptable
approaches to determine cleaning limits. The following
approaches may be considered as an alternative to the
MAC approach, which may have limited applicability for
biopharmaceutical products.
Cleaning Process Capability Approach
The cleaning process capability approach sets the accep-
tance limits for equipment cleaning based on demonstra-
tion that all carbon containing process materials have
been removed to the level that the cleaning process is
capable. The basis for the cleaning process capability
limit is that equipment surfaces cannot be cleaner than
the potential residual contribution from the last solution
of the cleaning process to contact equipment surfaces.
If TOC is used as the most suitable measure to demon-
strate removal of process material, the limit of process
capability of the cleaning process to measure cleanliness
would be based on the potential TOC contribution of the
final WFI rinse.
TOC results from surfaces that are below the clean-
ing process capability limit that cannot be differentiated
from TOC intrinsic to the final WFI rinse or from po-
tentially low levels of residual cleaning agent or process
material. TOC results that are above the cleaning process
capability limit would be as a result of residual clean-
ing agent or process material and not from the final
water rinse. It should be noted that this is a conserva-
tive approach to setting limits for equipment cleaning
verification and calculated limits are relatively low.
To calculate the TOC surface limit, the following
variables are required: equipment surface area, small-
est volume that the equipment could process (e.g.,
working volume), final rinse (WFI) TOC limit (source
of potential TOC contribution), and swab surface area.
It should be noted that the surface area and volume
are specific to the equipment to be cleaned and not to
the entire production train. When the MAC approach
is used, there is a concern of a cumulative carryover of
active product; which would not be removed through
common purification steps of subsequent product
production, which is the reason total surface area of all
equipment in the production train is used (1). Howev-
er, active product is not a concern once the product in-
activation and rinsibility are completed because active,
intact product would not be present after cleaning;
product fragments, just as other non-product proteins
(e.g., HCPs), would be removed during purification,
and product fragments created after cleaning are free
rinsing and easily removed from equipment surfaces.
The cleaning process capability limit may be deter-
mined for each piece of equipment, or the worst-case
piece of equipment in each production suite may be
used to set a limit to be used for all equipment in the
suite. The worst-case equipment will be the unit with
the largest surface area to volume ratio.
The following equation is used to calculate the
limit of TOC contribution on production equipment
surfaces that could be from the final WFI rinse. This
limit is determined by calculating the amount of TOC
on the equipment surface that would not result in TOC
concentration in minimum working volume allowed in
the equipment that would be greater than the accept-
able TOC limit of WFI (the final rinse source water):
Maximum Surface Residual TOC (ng TOC/cm2) =
To convert the Maximum Surface Residual TOC
limit into the limit for a swab sample, the following
equation is applied:
Residual TOC Swab Limit = Maximum Surface
Residual TOC (ng TOC/cm2) x SSA (cm2/swab) x 1 g
/1000 ng
Where:
Maximum Surface Residual TOC (ng TOC/cm2):
The maximum amount of residual material that is al-
lowed per square centimeter of production equipment.
SSA (cm2): Swabbed Surface Area, the area which
his swabbed for sample analysis. For example, 5 cm x
5 cm (2 inches x 2 inches) equals 25 cm2.
Unit Conversion (ng to g): converts units of ng to
g where 1 g equals 1000 ng.
Figure 1: Determination of Carryover Limit based
on Cleaning Process Capability.
An actual example of cleaning limit calculations
using the approach describe above is presented below
(Note: worst-case [tightest] limits will be calculated
where the production equipment surface area relative
Figure 1: illustrates the approach described above to calculate the residu-
al TOC limit as measured by a swab sample.

22 Special edition: Cleaning Validation


to working volume is large as is typically observed in
smaller equipment).
Example: 200 L Reactor (150 L minimum working
volume):
Maximum Surface
Residual TOC (ng TOC/cm2) =
= 2625 ng TOC/cm2
Residual TOC Swab Limit = 2625 ng TOC/cm2 x 25 cm2/
swab x 1 g /1000 ng
= 66 g TOC/swab
Acceptance limits for cleaning equipment set using
the Cleaning Process Capability approach is a conser-
vative limit that ensures removal of all carbon contain-
ing process residuals and cleaning agents to safe levels.
SAFETY FACTOR APPROACH
SAFETY FACTOR APPROACH
This approach is to determine the safety factor involved;
that is to calculate the reduction of the inactivated prod-
uct at the acceptance criteria level as an organic impurity
in the drug substance. This organic impurity limit is
0.10% (4), which is the equivalent to a Safety Factor of
1,000.
Concentration is the amount of active ingredient in
the drug substance/drug product.
Fifty-percent represents the approximate amount
of carbon in protein (5). This may also be calculated
based on the molecular makeup of the API if available.
The initial cleaning acceptance limits are typically
in the range of 1-10 ppm TOC for swab and rinse
samples. An example calculation is shown below; a 2
ppm acceptance limit with a product concentration of
100 mg/mL yields a Safety Factor of 25,000. Since this
is greater than a 1,000 Safety Factor, the 2 ppm accep-
tance limit has been appropriately set to demonstrate
adequate removal of residual active ingredient.
Another example calculation is shown below,
wherein a targeted Safety Factor of 10,000 (i.e., a 4-log
Rizwan Sharnez
reduction) is used to set the acceptance limit for a
product with a concentration of 100 mg/mL and a
molecular makeup of 53% carbon:
Once the initial acceptance limit has been set based
on the safety factor, the surface area limit can be cal-
culated:
Where:
The TOC acceptance limit is in ppm.
Volume is the amount of desorption solution used in
mL.
The surface area swabbed in cm2.
Continuing from the example above, the calculation
is shown below with a 5 ppm acceptance limit, 25 cm2
swab surface area, and 30 mL desorption solution:
Note: The Residual TOC Swab Limit is adjusted, as
necessary, based on surface area sampled where it is
not practical or possible to swab 25 cm2.
TOxICOLOGY THRESHOLD APPROACH
If it can be demonstrated that the biological products
becomes degraded and inactivated, application of a
toxicological threshold of concern (TTC) may be applied
in order to mitigate the risk of process residues (degraded
and inactivated fragments) affecting the next biophar-
maceutical produced (6-9). Once an appropriate TTC
has been determined based on structural class of process
residuals, a calculation such as the one below can be
applied.
Where:
ARL = Acceptable Residual Limit = g/cm2
TTC = Toxilogical Threshold of Concern = g/day
MBS = Minimum Batch Size for Subsequently Manu-
factured Product= g
Special edition: Cleaning Validation 23
Rizwan Sharnez
MDD = Maximum Daily Dose for Subsequently Manu-
factured Product = g/day
SA = Surface Area (SSA) = cm2
For example, degraded biopharmaceutical product
fragments may be considered to be Class I chemicals
with a residual soil threshold of 100 g/day. A 200 L
Final Product Vessel may have a surface area of 28,573
cm2: minimum batch size is 400 g, and maximum
daily dose is 50,000 g/day.
To calculate the TOC limit of a swab sample using
the ARL determined above, the following equation
would be used:
Residual TOC Swab Limit = Acceptable Residual
Limit (g/cm2) x SSA (cm2/swab) x 50%
Where:
Acceptable Residual Limit (g TOC/cm2): The maxi-
mum amount of residual material that is allowed per
square centimeter of production equipment.
SSA (cm2): Swabbed Surface Area, the area which his
swabbed for sample analysis. For example, 5 cm x 5
cm (2 inches x 2 inches) equals 25 cm2.
50%: Represents the approximate amount of carbon in
protein/protein fragments.
Continuing with example above to calculate the
ARL, the following is an example limit for Residual
TOC on a swab from production equipment:
Residual TOC Swab Limit = 28 g/cm2 x 25 cm x
(g TOC/swab) 50% TOC
= 350 g TOC/swab
If swab is desorbed in 40 mL of Low TOC Water,
the measured TOC limit as measured in ppb TOC
would be determined by dividing the Residual TOC
Swab Limit by 40 mL and converted to ppb using
the unit conversion of 1000 mL/L as described in the
equation below:
PERFORMANCE CONTROL LIMIT APPROACH
Performance Control Limits may be considered once
the cleaning validation studies have been completed and
routine cleaning consistently demonstrates the equip-
ment cleaning process removes process residue below
the acceptance limits, especially if the data is consider-
ably lower than the acceptance limit. The Performance
Control Limit approach does not change the level of
carryover that has previously been determined to be
acceptable, but it will establish a limit that is more reflec-
24 Special edition: Cleaning Validation
tive of the performance of the cleaning process. The
Performance Control Limit, sometimes referred to as an
Alert Limit, enables detection of a change in the perfor-
mance of the cleaning process and allows for a proactive
investigation into a potential cleaning process issue.
The Performance Control Limit approach discussed
below is based on the TOC data collected during on-
going cleaning studies. The evaluation of data should
be statistically based and strike an appropriate balance
between sensitivity to data shifts and excessive false
signals. Many standard statistical methods are based
on the assumption of normality and independence of
the data population. The setting of a control limit at
three standard deviations from the mean is an appro-
priate approach for setting a control (or performance)
limit, but it assumes a normally distributed dataset.
A control limit at three standard deviations from the
mean ensures a false out-of-tolerance (OOT) rate of
0.27%. This 0.27% value is referred to as the alpha
rate. The problem with the data typically generated
from effective cleaning processes is that the data are
not normally distributed, as shown in the following
example in Figure 2.
Because the data are not normally distributed, data
transformation techniques such as Box-Cox, mean
scores, reciprocal, negative binomial, etc. are to be
used to normalize data to apply appropriate statisti-
cal tools to establish an appropriate Performance limit
2 (10). The Box-Cox method is a log transformation that
optimizes the normality of the data set and was used
to transform the dataset presented above.
The Box-Cox method computes the lambda value to
optimize normality using the following equation:
Where Yoriginal is each TOC value, which must be > 0
Figure 2: Example Histogram of Cleaning Verification Data.

Figure 3: Box-Cox Transformation.
Figure 4: Effect of Using the Box-Cox Transformation.
Figure 5: Performance Control Limit from Example Dataset.
If the dataset contains an excessive number of zero
values, the 0 values should be removed and the alpha
rate (e.g., 0.27% or 0.0027) adjusted accordingly prior
to transforming the data with the Box-Cox method. In
the example dataset, 428 of 1034 results are 0. The
alpha rate (0.027) is therefore adjusted according to the
Rizwan Sharnez
number of 0 results relative to the total number of
results as described in the equation below:
After the review and adjustment for the 0 data
results, the Box-Cox transformation is performed us-
ing the adjusted alpha rate (in the example dataset, the
adjusted alpha rate is 0.0046). Figures 3 and 4, below,
depict the example TOC data that have been trans-
formed using the Box-Cox method.
The top-left histogram describes the distribution of
the original TOC dataset. This dataset is non-normal,
being truncated at zero. The same non-normal phe-
nomenon is displayed in the associated normal prob-
ability plot in the lower-left. The top-right histogram
describes the same data after applying the Box-Cox
transformation. In this case, the data are normally
distributed as evidenced with the normal probability
plot in the lower-right.
The Performance Limits are then back-calculated to
the original scale using the transformed dataset and
the equation below:
Yoriginal = (Ytransformed * lambda + 1)(1/lambda)
The Box-Cox transformed Performance Limit from
the example data is 4876 ppb TOC and is shown in
Figure 5.
Finally, as further cleaning studies are conducted,
additional TOC data will be collected. An appropri-
ate review of the overall dataset should be conducted,
and the performance limits adjusted if performance
changes for reasons that should be well understood.
CONCLUSION
Setting acceptable limits for process residue following
equipment cleaning in multiproduct biopharmaceutical
facilities requires an understanding of each products
composition and the effects of the cleaning process on
the API. The degrading and denaturing effects of chemi-
cal detergents should be studied for each product manu-
factured within the facility. Setting acceptance limits for
product carryover based on TOC can be accomplished
with the Cleaning Process Capability, Safety Factor, or
Toxicology Threshold approaches. As on-going cleaning
studies collect TOC data, these data can be evaluated
with the Performance Control Limit approach to ensure
control of the equipment cleaning process is maintained.
Special edition: Cleaning Validation 25
Rizwan Sharnez
ACKNOWLEDGEMENTS
We thank Kristina Conroy, Mariann Neverovitch,
Michael Hausladen of Bristol Myers Squibb, Rob Lynch
of GlaxoSmithKline, Jim Heimbach and Ben Locwin of
Lonza, Stephanie Donat, Gareth Sanderson of Novar-
tis, Martin Hammarstrm of Pfizer and David Bain of
BioPhorum Operations Group for their help and support.
ACRONYMS AND DEFINITIONS
Action Limit An empirical limit that the cleaning process
cannot exceed without potential impact to
product quality or patient safety.
PDE Permissible Daily Exposure (also called ADE,
Acceptable Daily Exposure) which represents
a dose of a drug to which a human may be
exposed per day or per dose (for biologics)
without any anticipated pharmacologic or
toxicological effects, so in the event of po-
tential carry-over of one API to another, there
would be no risk to the patient.
Alert Limit An empirical limit, statistically established
from study data, which is used to monitor
the quality of the cleaning process.
API Active Pharmaceutical Ingredient
Degrade To cause the cleavage and hydrolysis of
chemical bonds within peptides and amino
acid strings, such that the biological activity
is diminished or eliminated.
Denature To cause the tertiary structure of a biologi-
cal product to unfold, as with heat, alkali, or
acid, so that some of its original properties,
especially its biological activity, are dimin-
ished or eliminated.
MAC Maximum Allowable Carryover
Peptides A chemical compound containing two or
more amino acids (amino acid polymers) that
are coupled by a peptide bond. Peptides are
often classified according to the number of
26 Special edition: Cleaning Validation
amino acid residues. Oligopeptides have 10
or fewer amino acids. Molecules consisting
from 10 to 50 amino acids are called pep-
tides. The term protein describes molecules
with more than 50 amino acids.
TOC Total Organic Carbon
TTC Toxicological Threshold of Concern
REFERENCES
1. G.L. Fourman and M.V. Mullen, Determining Cleaning Valida-
tion Acceptance Limits for Pharmaceutical Manufacturing
Operations, Pharmaceutical Technology 17 (4), 54-60, 1993.
2. Methodology for Assessing Product Inactivation during Clean-
ing, Part I: Experimental Approach and Analytical Methods,
Journal of Validation Technology 17 (4), 2012.
3. Solubility of Proteins, Journal of Protein Chemistry 5 (6), 1986.
4. ICH, Q3A Impurities in New Drug Substances, 2008.
5. AR.C Beavis, Chemical mass of carbon in proteins, Analytical
Chemistry 65, 496-497, 1993.
6. Kroes et al., Structure-based thresholds of toxicological con-
cern (TTC): Guidance for Application to Substances Present
at Low Levels in the Diet, Food and Chemical Toxicology 42
65-83, 2004.
7. D.G. Dolan, B.D. Naumann, E.V. Sargent, A. Maier, and M.
Dourson, Application of the Threshold of Toxicological Con-
cern Concept to Pharmaceutical Manufacturing Operations,
Regulatory Toxicology and Pharmacology 43, 19, 2005.
8. J.P. Bercu and D.G. Dolan, Application of the Threshold of
Toxicological Concern Concept When Applied to Pharma-
ceutical Manufacturing Operations Intended for Short-term
Clinical Trials, Regulatory Toxicology and Pharmacology 65,
162167, 2013.
9. ICH, Assessment and control of DNA reactive (mutagenic) impurities
in pharmaceuticals to limit potential carcinogenic risk. M7 Step 2
version (2013).
10. Statistics for Experimenters: Design, Innovation, and Discovery, 2nd
ed., 2005.
 Tim Sandle

Aseptic Transfer Risk

Assessment:
A Case Study | IVT
Tim Sandle, Ph.D.

ABSTRACT
This paper uses one example of a risk assessment approach to illustrate
how risk assessment can be incorporated into good manufacturing
controls. The specific activity assessed involves transferring a set of
sterilised stoppers from an autoclave to a filling machine within a ster-
ile manufacturing facility. The risk assessment approach adopted is a
form of HACCP (Hazard Analysis Critical Control Points). Approaches
to risk assessments are discussed. Key activities are described. Docu-
mentation is critical. There is no such thing as zero risk. A deci-
sion is thus required as to what is acceptable risk. Key components
of HACCP including hazard analysis and critical control points. The
seven pillars of HACCP are described. Stepwise activities needed to
accomplish the risk assessment are discussed. This case study de-
scribed a low risk activity. Rationale for the assessment is described.
This discussion described a risk assessment approach using a relatively
simple case to illustrate its potential for other applications.

INTRODUCTION
Risk analysis and risk management are significant considerations in
current pharmaceutical manufacturing practices. All areas and func-
tions in the pharmaceutical manufacturing plant assess level of risk to a
process and then take steps to eliminate that risk. This paper uses one
example of a risk assessment approach to illustrate how risk assessment
can be incorporated into good manufacturing controls.
Risk assessment involves identifying risk scenarios either prospectively
or retrospectively. The former involves determining what can go wrong
in the system and all the associated consequences and likelihoods; the
latter this looks at what has gone wrong. Risk assessment is then used to
assess the process, product, or environmental risk and to aid in formulat-
ing the appropriate actions to prevent the incident from re-occurring (1).
The case examined here is a prospective risk assessment.
The case study involves transferring a set of sterilised stoppers from
an autoclave to a filling machine within a sterile manufacturing facility.
The risk assessment approach adopted is a form of HACCP (Hazard
Analysis Critical Control Points).

APPROACHES TO RISK ASSESSMENT

There are various approaches to risk assessment being used in the phar-
maceutical industry. Each has their respective merits. The use of risk
assessment is commonly expected by regulatory authorities (2).
There are various tools that can be drawn upon for conducting risk
assessments. Some common methods include FMEA (Failure Mode
and Effects Analysis), FTA (Fault Tree Analysis), and HACCP (Hazard
Analysis Critical Control Points). Many of these employ a scoring ap-
proach. No definitive method exists for all applications and different

Special edition: Cleaning Validation 27

Tim Sandle
approaches are useful for different situations (3).
The various approaches differ in their structure
and with the degree of complexity involved. None-
theless, there are some broad similarities. The differ-
ent analytical tools are similar in that they generally
involve:
Constructing diagrams of work flows,
Pin-pointing areas of greatest risk,
Examining potential sources of contamination,
Deciding on the most appropriate sample meth-
ods,
Helping to establish alert and action levels,
Taking into account changes to the work process
/ seasonal activities.
Documenting risk assessments is important.
When a risk assessment is presented to an auditor,
it is likely that the reviewer will be concerned with
whether the risk assessment is traceable to a risk
methodology and that the process is clear, under-
standable, and that it has been performed consistent-
ly. In this sense, the reviewer will want to under-
stand how the decisions were made and that the risk
(or problem) was defined upfront. With the question
established, the reviewer will also wish to see if the
process performed actually answered the question in
a meaningful way using scientific principles.
Important points to remember for any risk assess-
ment are the following:
There is no such thing as zero risk. A decision pharmaceutical processes (5).
is thus required as to what is acceptable risk.
Risk Assessment is not an exact science - differ-
ent people will have different perspectives on the
same hazard.
Risk assessments should always:
Be based on systematic identifications of possible
risk factors
Take full account of current scientific knowledge
Be conducted by people with experience in the
risk assessment process and the process being
risk assessed
Use factual evidence supported by expert assess-
ment to reach conclusions
Do not include any unjustified assumptions
Identify all reasonably expected risks-simply and
clearly, along with a factual assessment and miti-
gation where required
Be documented to an appropriate level and con-
trolled/approved
Ultimately be linked to the protection of the
patient
Should contain objective risk mitigation plan.
28 Special edition: Cleaning Validation
Before embarking upon risk assessment it is impor-
tant to establish and to define:
Develop and agree on the risk question. A clearly
defining the risk question facilitates selection
of the appropriate risk assessment tool, identi-
fies relevant data, information and assumptions;
assists in the identification of resources, respon-
sibilities and accountabilities; and ensures that
appropriate focus on the business objective is
maintained.
Select the most appropriate risk assessment tool.
The selected risk assessment method or tool will
be used to organise collected data, understand
what steps can be taken to reduce or control risk,
and to help make appropriate decisions.
APPLICATIONS TO CLEANROOM ENVIRONMENTS AND
PHARMACEUTICAL PROCESSES
The primary risk assessment approaches can al-
low for a complete review of operations within the
cleanroom ensuring those facilities, operations and
practices are also satisfactory. The approaches recog-
nise a risk, rate the level of the risk, and then develop
a plan to minimise, control, and monitor the risk.
For example, the monitoring of the risk will help
to determine the frequency, locations, and level for
environmental monitoring (4).
This case study outlined in this paper uses a
modification of the HACCP method, which is some-
times called the Lifecycle Approach. The approach
adopted has been used for similar assessments of
There are numerous risk factors to be considered
when analysing aseptic processing. The HACCP
approach can help to identify these risk factors. The
following factors shown in Figure 1 are useful to note
Figure 1: ASEPTIC PROCESSING RISKS

when undertaking HACCP analysis in pharmaceuti-
cal manufacturing.
HACCP
HACCP was developed during the 1960s from
collaboration between NASA, a food company (the
Pillsbury Company), and the US Army Natick Labo-
ratories. The objective was to provide a zero-defect
food supply for astronauts. HACCP was derived from
Failure Mode Analysis (6). There are two key compo-
nents of HACCP:
Hazard Analysis: Determining what microbio-
logical, physical, or chemical risks are associated
with a process.
Critical Control Point: A point, step, or procedure
at which control can be applied.
HACCP generally involves an assessment of the fol-
lowing conditions known as the seven pillars.
1. Conducting a hazard analysis. List all potential
hazards associated with each step, conduct a
hazard analysis, and consider any measures to
control identified hazards.
2. Determining the Critical Control Points (CCPs).
Critical control points have to be defined as
processing steps at which necessary action can
be applied to ensure and maintain compliance
with specified conditions. Those points are
identified with appropriate measures that can
be applied to control each hazard. If no such
critical control points can be established, prod-
uct specific validation should eliminate poten-
tial risks of a certain process step.
3. Establishing critical limit(s). Establish critical
limits for each CCP.
4. Establishing a system to monitor control of the
CCP.
5. Establishing the corrective action to be taken
when monitoring indicates that a particular
CCP is not under control.
6. Establishing procedures for verification to con-
firm that the HACCP system is working effec-
tively.
7. Establishing documentation and record keep-
ing.
Aspects of these have been utilized in the case study.
In general, the advantages of the HACCP ap-
proach are that it allows for a systematic overview
of the process for the evaluation of each process-
ing step, allows each step to be examined the
possible risks, and allows for the specification of
measures required for controlling each risk. The
Tim Sandle
primary disadvantage of HACCP is that, unlike
FMEA, HACCP cannot be used to rank or priori-
tize risks.
DECONSTRUCTING THE PROCESS
Four stepwise activities are needed to accomplish the
risk assessment:
A route map. The facility is drawn and the route
indicated
Identification of hazards, which can be divided
into biological; physical; equipment; transport
and chemical. This will allow an assessment of
existing control measures
Process flow
Assessment of environmental monitoring. This
will determine if the activity is safe to proceed (7).
Route Map
The first step was to outline a route map. This
focused on transporting the stoppers by the shortest
and safest route possible as illustrated in figure 2.
Figure 2: ROUTE MAP. Arrows indicate stopper transfer route from auto-
claves to filling room
Identification of Hazards
The second step was to understand the hazards and
the process. The primary hazards with an aseptic
process are biological, physical, and equipment re-
lated (8) as displayed in Table 1.
Process flow
The third step was to outline the process flow as
shown in Figure 3.
Environmental monitoring
The fourth step involved assessing dynamic state
viable microbiological environmental monitoring
and particle counts. If data have been satisfactory
and there are no indications of adverse trends, the
proposal can be advanced. If there is a level of
risk, this must be addressed first using appropriate
Special edition: Cleaning Validation 29
Tim Sandle

Table 1a: IDENTIFICATION OF HAZARDS

Figure 3a: Process flow with CCPs (critical control points) marked.
Process flow identifies hazards.

Table 1b: IDENTIFICATION OF HAZARDS

corrective and preventative action. Undertaking an

unusual event in a high-risk situation will com- Figure 3b: Process flow with CCPs (critical control points) marked.
pound the problem. Process flow identifies hazards.

30 Special edition: Cleaning Validation


RISK ASSESSMENT PERFORM A SIMULATION
Before undertaking the activity, a simulation should
be performed so that any previously unforeseen prob-
lems can be noted and further preventative measures
taken. If any variability is expected, the simulation
should be repeated.
The simulation should be timed and the number
of staff required noted. The focus should remain on
the ease of transit. In this case study, the duration
of the activity is five minutes and the number of staff
involved is two.
CONCLUSION
From three possible options -- high risk, medium risk
or low risk -- the conclusion of this risk analysis was
that the risk level was low. This evaluation is based
on the successful identification of the hazards and
control points. The strongest evidence for this came
from the following:
The process being performed by trained clean
room personnel operating in an aseptic manner
The autoclaved stoppers being contained within
a sealed box. The seal remains intact throughout
the transfer
The outlet at the bottom of the box has an integ-
rity tested vent filter
An additional vent filter is placed on the top of
the box
The stoppers are wheeled on a trolley which
minimises human intervention
The microbiological environmental monitoring
for the rooms is satisfactory.
It is important to note the impact of change and
to review any risk assessments on a regular basis in
developing any risk assessment.
This case study may or may not be typical. It may
or may not be wise for an organisation to undertake
such an activity. The purpose of this discussion was
to demonstrate an example of the risk assessment ap-
proach using a relatively simple case so that the wider
application can be appreciated.
Tim Sandle
REFERENCES
1. Sandle, T. (2011): Risk Management in Pharmaceutical
Microbiology. In Saghee, M.R., Sandle, T. and Tidswell, E.C.
(Eds.) Microbiology and Sterility Assurance in Pharmaceuticals
and Medical Devices, New Delhi: Business Horizons, pp553-
588
2. Sandle, T. and Lamba, S. S. Effectively Incorporating Quality
Risk Management into Quality Systems. In Saghee, M.R.
(2012) Achieving Quality and Compliance Excellence in Pharma-
ceuticals: A Master Class GMP Guide, New Delhi: Business
Horizons, pp89-128
3. Sandle, T. (2013). Contamination Control Risk Assessment.
In Masden, R.E. and Moldenhauer, J. (Eds.) Contamination
Control in Healthcare Product Manufacturing, Volume 1, DHI
Publishing, River Grove: USA, pp423-474
4. Sandle, T. (2003): The use of a risk assessment in the phar-
maceutical industry the application of FMEA to a sterility
testing isolator: a case study, European Journal of Parenteral and
Pharmaceutical Sciences, 8(2): 43-49
5. Munro, M. J., Millar, B. W. and Radley. A. S. (2003):
A Risk Assessment of the Preparation of Parenteral
Medicines in Clinical Areas, Hospital Pharmacist, Vol. 10,
pp303-305
6. Gavin, A., and Weddig, L.M. (Eds.). (1995): Canned Foods:
Principles of Thermal Process Control, Acidification and Con-
tainer Closure Evaluation, Food Processors Institute, Washing-
ton, D.C., pp5-10
7. Sandle, T. (2006) Environmental Monitoring Risk Assess-
ment, Journal of GXP Compliance, 10(2): 54-73
8. Whyte, W. and Eaton, T. (2004): Assessing microbial risk
to patients from aseptically manufactured pharmaceuticals,
European Journal of Parenteral and Pharmaceutical Sciences, 9
(3): 71-79
9. Whyte, W. and Eaton, T. (2004): Microbiological contamina-
tion models for use in risk assessment during pharmaceutical
production, European Journal of Parenteral and Pharmaceutical
Sciences, 9 (1): 11-15
10. Sandle, T. (2004): General Considerations for the Risk Assess-
ment of Isolators used for Aseptic Processes, Pharmaceutical
Manufacturing and Packaging Sourcer, Samedan Ltd, Winter
2004, pp43-47
Special edition: Cleaning Validation 31
Tim Sandle

People in Cleanrooms:
Understanding and Monitoring
the Personnel Factor | IVT
Tim Sandle, Ph.D.

ABSTRACT
Cleanroom contamination can arise from a number of sources. Most
contamination within the pharmaceutical facility can be traced to
humans working in cleanrooms. The paper discusses staff gowning and
personnel behavior in pharmaceutical cleanrooms, and how cleanroom
risk can be minimized. The human skin ecosystem is discussed. The
Human Microbiome Project (HMP) from the US NIH characterized
microorganisms found in association with both healthy and diseased
humans. Information from this project has great impact on cleanroom
activities including gowning practices. Topics associated with clean-
room garments are discussed including fabric types, garment lifespan,
recycling, laundering, human changing procedures, training, behavior,
hand sanitization, ongoing assessments, and associated topics.

INTRODUCTION
Cleanroom contamination can arise from a number of sources. These
may vary depending upon the type of cleanroom, its geographic loca-
tion, the types of products processed, and so on. Nevertheless, these
sources can generally be divided into the following groups1:

People
Water
Air and ventilation
Surfaces
Transport of items in and out of clean areas

Most contamination within the pharmaceutical facility can be traced to

humans working in cleanrooms2. This is, in some way, evidenced from
the association of microorganisms transient or residential to skin being
the primary isolates from environmental monitoring in controlled envi-
ronments3. Human personnel shed high numbers of skin cells mostly as
skin flakes. The cleanroom garments worn by personnel cannot contain
all human detritus.
The paper discusses staff gowning and personnel behavior in phar-
maceutical cleanrooms. Further, it considers how cleanroom risk can
be minimized. Basic training for all cleanroom staff including activities
such as cleanroom entry and gowning practices is examined.

HUMAN SKIN
Before proceeding to look at gowning, it is worthwhile to consider
the human skin ecosystem. The human body is an intricate system
that hosts trillions of microbial cells across the epithelial surface and
within the mouth and gut. These microorganisms play a role in human
physiology and organ function including digestion and immunity. The
microorganisms also affect the outside environment as they are shed

32 Special edition: Cleaning Validation


AREA NUMBER OF MICRO-
ORGANISMS/cm2
Scalp 1 million
Saliva and nasal fluid 10 million/gram
Back 100
Groin 1 20 million
Forehead 100 1000
Hand 10,000 100,000
Feet 1 million
Table 1: HUMAN BODY SITES AND TYPICAL NUMBERS OF
MICROORGANISMS WITHIN AREA
from the skin or deposited through different orifices.
This latter issue has important implications for clean-
rooms. The outer layer of human skin can host up to
one million microorganisms per square centimeter4.
The population, as well as the diversity, varies ac-
cording to anatomical locale.
Research suggests that a typical person sheds
1,000,000,000 skin cells per day of a size 33 m x
44 m x 4 m -- equivalent to a rate of 30,000 to
40,000 dead skin cells shed from the surface of the
skin every minute. Of these, Whyte indicates that ap-
proximately 10% of particles carry microorganisms5.
There are, on average, four microorganisms per skin
cell. A term commonly used to describe skin flakes
with adhered microorganisms is microbial carrying
particles.
The significance of this is that people are not only
a source of contamination, but also are an agent for
transferring contamination possibly to locations that
could pose a product risk. Microorganisms are spread
from sneezing, coughing, and touching. While micro-
organisms suspended in the air are less of a concern,
should such organisms gravitate towards a product or
critical location, they may present a significant risk.
HUMAN MICROBIOMe PROjeCT
Our understanding of the risk the people pose to
cleanrooms has been advanced by the findings from
the Human Microbiome Project. The Human Microbi-
ome Project (HMP) is a United States National Insti-
tutes of Health initiative. The project goal is to iden-
tify and characterize the microorganisms found in
association with both healthy and diseased humans
(the human microbiome). The human microbiome
describes the aggregate of microorganisms and their
genetic interactions that reside on the surface and in
deep layers of skin, in the saliva and oral mucosa, in
the conjunctiva, and in the gastrointestinal tract.
Some of the most illuminating HMP research has
been with the human skin. The skin is a complex
ecosystem, supporting a range of microbial commu-
nities that live in distinct niches. These niches are
Tim Sandle
affected by available nutrients as well as by several
non-nutritional factors such as pH, humidity, and
temperature. With the skin, however, as epithelial
cells are continually being shed, many microbial
communities on the external surface are rarely
stable6.
The outcomes of the HMP research have shown
that there is a high population on, and a considerable
diversity of microbial species across, the outer layer
of the skin. There are approximately 1000 species
of bacteria from 19 phyla on human skin. Of these,
most bacteria can be categorized into four phyla:
Actinobacteria (51.8%). Actinobacteria are a
group of Gram-positive bacteria with high gua-
nine and cytosine content, such as Micrococcus,
Corynebacteria and Propionibacteria.
Firmicutes (24.4%). This includes the genera
Clostridia and Bacillus.
Proteobacteria (16.5%). This is a major phylum of
bacteria that includes a wide variety of pathogens
such as Escherichia, Salmonella, Vibrio, Helico-
bacter, and many other notable genera.
Bacteroidetes (6.3%). The phylum Bacteroidetes
is composed of three large classes of Gram-
negative, nonspore-forming, anaerobic, and rod-
shaped bacteria7.
Reasons for the topographical variations relate to
the physicochemical properties of the skin. This is
especially so for temperature, pH, amounts of oil, and
moisture8. From this, there are three main ecological
areas of the skin: sebaceous, moist, and dry. Exam-
ples of microbial divergence include Propionibacteria
and Staphylococci species dominating the sebaceous
areas (with a high oil content). Dry, calloused areas
(arms and legs), Gram-positive cocci (primarily the
Micrococcaceae) are found on the arms and legs and
Gram-positive rods are found in high numbers on the
torso. Staphylococci and Corynebacteria are found
together with some Gram-negative bacteria in moist
areas9. These types of microorganisms generally
reflect the types recovered from cleanrooms10.
The reason that Gram-positive bacteria predomi-
nate across the skin is because the skin is generally a
dry environment, and any fluids present on the sur-
face generally have a high osmotic pressure11. Thus
Gram-positive bacteria (especially the Staphylococci
and Micrococci) are better adapted for such environ-
ments, not least to being resistant to desiccation.
Where other species occur this is due to variations in
temperature and with areas of higher sweat produc-
tion12. For example, this can lead to higher levels
of fungi on the feet13. In relation to pharmaceutical
manufacturing, the presence of any such organisms
remains problematic.
Special edition: Cleaning Validation 33
Tim Sandle
A further observation is that the ratio of the micro-
organisms recovered from the skin is relatively evenly
divided between the aerobic and the anaerobic. The
aerobic microorganisms tend to live on the outermost
layers of the skin and the anaerobic microorganisms
live in the deeper layers of the skin and hair follicles14.
The information from the human microbiome proj-
ect about the rich depth of variety of microorganisms
on the skin introduces several implications for clean-
room environmental monitoring. The most important
question of whether gowning practices are adequate
to exclude all microorganisms from the richest areas
of the skin microbiome. This is a pertinent point
given that most bacteria free-floating in cleanroom air
current are not free-living but are instead the result
of direct particle shedding of desquamated skin cells
and subsequent re-suspension of skin detritus in the
air stream.
The answer to this question should lead to a consid-
eration of:
1. The types of cleanroom undergarments used
and an examination as to whether these pro-
vide an effective barrier, especially for the more
moist parts of the body.
2. The importance of the outer gown covering all
parts of the body, including the forehead.
3. The quality of cleanroom certified undergar-
ments.
4. The level of training required for operators in
relation to gowning and the way that gowning
qualification as conducted.
5. How long a cleanroom suit should be worn for
in relation to material integrity against operator
perspiration.
6. The environment in which gowns are donned,
where higher air-change rates might prove effec-
tive.
7. How often gowns should be recycled which
involves washing and irradiation. At some point
the material fibers will weaken, thereby reduc-
ing the bacteria filter efficiency of the gown.
The wearer of the gown should know what
types of testing is conducted on recycled gowns
and what the procedures are for rejecting gowns
where a loss of integrity is detected.
Cleanroom microbiologists may wish to consider
how concerned they are with each of the items listed
in excluding microorganisms found on all regions of
the skin. There must be good understanding of the
environmental monitoring methods used to assess
cleanrooms. These may not show how good or bad
gown changing and gown wearing is. These concerns
are best addressed through good gowning practices.
34 Special edition: Cleaning Validation
PROTeCTING PRODUCTS FROM PeOPLe
Despite some advances with automation and robotics,
in most situations people cannot be eliminated from
cleanrooms. Control of contamination from people in
cleanrooms is achieved by application of two prin-
ciples:
We wrap the people to minimize the amount of
shedding of microorganisms.
We put localized protection around the prod-
uct to minimize the amount of contact with the
people.
The localized protection issue is achieved through
local air protection, such as unidirectional airflow
cabinets and isolators. With clothing, personnel
working in cleanrooms are required to wear special
clothing designed for the clean environments. Such
clothing is necessary, as indicated above, because the
human body creates its own micro-environment of
potentially damaging particulate contamination.
To be effective, cleanroom clothing must:
Form a particulate barrier for the human micro-
environment.
Allow freedom of movement and be comfortable.
Address any specialist requirement, e.g. static
dissipation.
Avoid being a significant particulate contributor
in itself.
Cleanroom garments must meet specific protection
criteria. Not all cleanroom garments are of the same
quality. This involves manufacturing the garments
from special materials, following particular construc-
tion methods, and then tailored for individual styling.
The gowns must be comfortable, easy to apply and
practical in use. Some gowns are disposable and
others are made to be re-laundered and sterilized
depending on the cleanroom grade.
CLeANROOM GARMeNTS AND FABRICS
Fabrics used in the manufacture of cleanroom gar-
ments must have the following features15:
Be low in particulate shedding.
Permit the body to breathe while trapping parti-
cles within the garment. The contaminant should
be retained within the garment and not released
into the surrounding atmosphere.
Be flexible enough for comfortable wearing.
Withstand repeated cleaning and sterilization
cycles.
Meet any specific requirements such as control of
static.

Meet opacity requirements.
Look and feel as good as possible.
Be cost-effective.
Fabric Categories
There are three broad categories of fabric used in the
construction of cleanroom garments:
Woven fabrics. Woven or re-usable fabrics are
the most commonly used fabrics in cleanroom
environments. Such garments are woven on
sophisticated looms from yarns of continuous
filaments of polyester. The thickness of the yarn
and filaments is important -- the finer the yarn,
the tighter the weave can be made, and the bet-
ter the filtration. The pattern and tightness of the
weave is important to reduce the pore size to a
minimum. The use of continuous filament poly-
ester means that there are few loose ends from
which particles may be shed.
Laminated or membrane fabrics. Laminated
fabrics are favored for some high-grade micro-
electronic environments. These types of garments
are not commonly used in the pharmaceutical
sector.
Disposable or limited life materials. The most
common of these non-woven fabrics are from
spun bonded olefin and polypropylene. Compris-
ing a densely interlinked matt of fibers, these
fabrics can provide good results for a limited
period. Garments from such materials need to be
processed and decontaminated before use in the
cleanroom. Disposable or limited use garments
are mainly used in those environments where
protection of the wearer against potentially haz-
ardous products is required.
Garment Considerations
Garments are designed to provide protection for the
head, body, hands and feet. In establishing a system
for garment selection, it is important to consider the
broader aspects of cleanroom use: suitability of fabric,
garment style, layers, the nature of the tasks involved,
costs, regulatory requirements, and any specific cus-
tomer requirements. The classification of the clean-
room will inevitably be the major factor in determin-
ing the degree of personnel protection required and
the fundamental choice of garments.
One important issue with gowns is the maximum
length of time that a gown can be worn. As people
perspire, the integrity of the gown will weaken. Com-
plicating factors are the temperature and humidity
of the cleanroom and the variations between people.
The length of time will also depend upon the grade
of the cleanroom. In aseptic areas, such as ISO 14644
class 7 / EU GMP Grade B areas, gowns are typically
Tim Sandle
worn only for the length of the shift (normally four
hour periods to enable operators to take breaks). In
lower grade cleanrooms, a gown might be worn for
several sessions during the course of the working day.
Other factors affecting the lifespan of the gowns
that are subject to recycling are repairs and the num-
ber of permitted washing cycles. With repairs, it is
prudent to have a repair policy. This will vary across
facilities, and again, it will be affected by the clean-
room class. With aseptic areas, if a gown becomes
torn it is normally discarded. In other grades of
cleanroom, a gown can be repaired depending upon
the size of the hole and the impact on the material.
Some organizations set a maximum size for any hole
or tear and for the number of times a gown can be
repaired.
Gowns that are recycled are subject to laundering.
Gowns are washed by special washing machines with
suitable detergents, dried, folded, and then wrapped
in cleanroom packaging. For gowns that are to be
used in aseptic areas, such gowns are irradiated. A
policy should be in place outlining how often a gown
can be processed -- typical times range between 20
and 40 times. To make the tracking task easier, many
gowns sterilized by irradiation or gassing are fitted
with barcodes and scanned. It is further important
to establish the extent that the sterilization process
affects the integrity of the gown material.
In order to assess the contamination risks from
re-laundering, gowns are subject to particle count-
ing. There are different ways to do this, although the
most common means is the Helmke Drum particle
emission test. With this, the test method simulates
particle shedding of clothing under movement. The
garment under test is tumbled in a rotating drum
(approximately 10 revolutions per minute) to release
particles from the surface of the cleanroom garment
in a controlled manner. An automatic particle counter
is used to sample the air within the drum to deter-
mine the average particle concentration of the air
during the initial ten minutes of the test. The com-
mon standard is the IEST Recommended Practice
RP-CC003.3: Garment System Considerations for
Cleanrooms and Other Controlled Environments.
An alternative measure is the Body Box test. This
method simulates particle filtration and release under
real wear conditions. As a consequence it measures
the contamination of the cleanroom by the cloth-
ing/wearer. For this, particle counters determine the
quantity of particles generated by the wearer/garment
that are emitted into the chamber.
CHANGe PROCeDURe
Cosmetics, such as powder, rouge, eye liner, mascara,
and lipstick must be banned in cleanroom environ-
ments. Jewelry, such as rings, watches, necklaces,
Special edition: Cleaning Validation 35
Tim Sandle
bracelets, earrings and other items, together with all
forms of visible piercing, are commonly not allowed
in cleanrooms.
The best method of changing into cleanroom gar-
ments is one that minimizes contamination getting
onto the outside of the garments. Change areas can
vary in design, but it is common to find them divided
into three zones:
1. Pre-change zone. Outside of changing rooms
tacky mats or polymeric flooring can be posi-
tioned to help reduce the level of particles car-
ried on footwear16.
2. Changing zone. The changing room design
contributes to the assurance of appropriate
personnel access and microbial contamination
control. The changing room should be provided
with filtered air. Intermediate (bag) filters will
typically be suitable for this purpose, though
High Efficiency Particulate Air (HEPA) filtration
may be used. The air pressure should be nega-
tive with regards to the manufacturing area cor-
ridor, but positive relative to external adjacent
areas.
3. Cleanroom entrance zone. This must be of the
same grade or class as the main cleanroom into
which the area leads.
Ideally there should be separate routes through
airlocks for material required in cleanrooms. Tak-
ing items through personnel change areas should be
discouraged.
TRAINING
Personnel training in gowning is an important func-
tion. Gowning practices must be assessed periodi-
cally and monitored frequently. Training programs
should ideally include visual assessment and microbi-
ological assessment. The microbiological assessment
varies, but can include the exposure of settle plates
during the change process and the assessment of
gown cleanliness through post-use suit contact plates.
The results of the cleanroom sampling should not ex-
ceed those of the room class. If results are exceeded,
the individual may be an unusually high shedder of
skin particles.
Training required for staff who work in cleanrooms
should include:
Introduction to micro-organisms and microbio-
logical contamination control.
Entry and exit of production facilities (including
gowning).
Personal hygiene training.
Microbiological risks associated with specific
production tasks.
36 Special edition: Cleaning Validation
Training must be documented and regularly
reviewed. Training must be effective. Actual perfor-
mance of personnel competency in gowning should
be demonstrated on a regular basis.
CLeANROOM PeRSONNeL BeHAVIOR
Working in clean environments demands knowledge,
discipline, motivation as well as a thorough under-
standing of contamination risks among all personnel
involved. Each individual cleanroom should have its
own documented rules and procedures.
Training includes reminding personnel that they
must not be allowed to touch critical products and
equipment with their naked hands. All critical work
must be undertaken wearing gloves. Critical ac-
tivities requiring personnel contact such as aseptic
processing or sampling must be done through the
use of clean utensils such as tweezers, forceps, and
the equivalent. All devices and gloves used must
fully comply with the cleanliness demands of the
cleanroom and work undertaken in the cleanroom.
They must be cleaned, disinfected, or sterilized as
appropriate for the criticality or activity and risk of
contamination.
Another aspect of best practice is in instruct-
ing personnel in the appropriate behaviors within
the cleanroom. The generation of contamination is
proportional to activity conducted. A person with
head, arms, and body moving can generate about
1,000,000 particles 0.5 m/min. A person who is
walking can generate about 5,000,000 particles 0.5
m/min. However a person in motionless position
can generate only 100,000 particles 0.5 m/min.
In addition, personnel should reduce activities like
talking, singing, whistling, coughing, sneezing etc.,
especially when being close to the handled products
and production equipment.
People working in cleanrooms and other forms of
controlled environments must be physically healthy.
Diseases in the upper respiratory tract as well as
stomach disorders can create problem in hygienic
applications.
Another factor that can impact upon the environ-
ment is the number of people in the cleanroom. Only
necessary and limited number of persons should be
allowed in a cleanroom at the same time. The more
persons simultaneously present in a cleanroom, then
the higher the contamination level will be, i.e., the
higher concentration of particles in the air). This is
particularly important in relation to changing rooms.
HAND SANITIZATION
Good personal hygiene is a requirement of all phar-
maceutical cleanroom activities. However, studies
show poor compliance is common in relation to basic
hand washing technique. Hand hygiene and glove

Figure 1: HAND SANITIZATION (IMAGE: TIM SANDLE)
hygiene are important given the high numbers of
microorganisms found on the human body including
the hands and the risks posed by hands as a means
of contamination transfer. Microorganisms associ-
ated with hands are found mainly on the surface of
the skin and under the superficial cells of the stratum
corneum. The dominant species is Staphylococcus
epidermidis that is found on almost every hand,
together with other species of Staphylococcus and
species of the genera Micrococcus17.
Hands must be washed with soap and water prior
to entry to the cleanroom. Hand washing facilities
should not be located in an actual cleanroom, but
rather in an area leading to the cleanroom changing
room. As an alternative, a hygienic handrub can be
used.
Where gloves are required these should be put on
using a method designed to prevent the ungloved
hand from touching the clean or sterile outer part
of the glove. Once in the cleanroom, gloved hands
should be subject to periodic hand sanitization18.
When decontaminating hands with an alcohol-
based antiseptic hand rub, apply product to palm
of one hand and rub hands together, covering all
surfaces of hands, fingers and wrists, until hands are
dry (alcohol-based hand rubs are not to be used with
water). The process typically takes between thirty
seconds and one minute. Follow the manufacturers
recommendations regarding the volume of product to
use.
The technique for applying alcohol to gloved hands
is similar to applying a handrub to skin. It is im-
portant to ensure that all surfaces are covered. With
glove sanitization, there are two alcohols of choice:
ethyl alcohol (ethanol) and isopropyl alcohol (IPA).
Other alcohols, such as methyl alcohol (methanol) are
unsuitable19. Of the two alcohol forms, IPA is slightly
more bactericidal than ethanol, although ethanol has
better viricidal properties20. Another factor is appli-
cation to the skin, and here IPA can be quite harsh.
Tim Sandle
Thus, ethanol is more often applied to bare skin
(often in a denatured form) whereas IPA is more often
applied to gloves.
These sanitizers have bactericidal action against
vegetative cells but not spores. The concentration of
alcohol to water varies, although the optimal range is
60 to 90% (volume/volume). Below 60%, bactericidal
action drops, and above 90% there is insufficient wa-
ter for the bacterial cell to absorb water. The alcohol
does not enter the cell and is unable to denature the
bacterial proteins21. Most preparatory concentrations
are 70%.
While most bacteria are killed after ten seconds of
contact with alcohol22, contact times in practice are
longer due to the variability of hand rubbing. Typical
contact times are thirty seconds.
It is important that the selection of a hand sani-
tizer is qualified. There are different approaches that
can be taken for qualification. Most of these require
individuals to wear gloves and to place their hands
into broth containing a high concentration of a non-
pathogenic microorganism. Disinfectant is then ap-
plied, and the bacterial reduction is assessed through
placing the treated hands into broth and performing
dilutions.
ON-GOING ASSeSSMeNT
In higher-grade cleanrooms such as those used for
aseptic processing, it is common practice to assess the
risk of personnel to the process by taking suit contact
plates of the gown as worn by the person as they
leave the aseptic area. The gown must be discarded
after the plates have been taken due to the potential
effect on the gown integrity when an agar plate con-
tacts the gown.
It is good practice to begin suit sampling with
a higher number of samples. These can then be
reduced over time. Some facilities perform more
samples from the gown during gowning test qualifi-
cations compared with routine sampling. Sites con-
sidered for selection include the top of the head, the
face mask, both arms, middle torso, and both legs.
In terms of limits, for EU GMP Grade B/ISO class 7
areas, the aim is often to adopt the same limits as per
the limits applied to finger plates. The action level for
gowns is ordinarily 5 CU/25cm 2.
Experience has shown that higher counts are
obtained from the top of the head, perhaps because
this is the warmest region of the body. Care must be
undertaken when sampling as so not to break the
integrity of the gown.
In addition to gowning control, a procedure should
be in place for the notification of health conditions
by staff. Staff who are ill (coughs, colds, and so on)
should not enter cleanrooms. This is because the
Special edition: Cleaning Validation 37
Tim Sandle
illness may affect product quality. It is important to
control the potential risks from personnel carrying
Infectious disease.
Open lesions on any exposed part of body.
Shedding skin conditions, such as eczema or pso-
riasis, dermatitis, and dandruff (skin scales may
harbor objectionable micro-organisms that may
impact pharmaceutical products and patients).
Gastric upsets.
Personnel with any of the above conditions must be
excluded from working within cleanrooms for the
duration of their illness.
CONCLUSION
This paper has considered the personnel factor and
the relationship between people and cleanrooms. It
addressed why people are a risk in relation to the
skin microbiome, and how good gowning practices
can help to minimize that risk. The paper has also
considered other factors that can affect contamination
risks from people, including the importance of good
behaviors and the necessity of hand sanitization.
Capturing these various issues through procedures
and imparting the key concepts through training are
a necessary part of cleanroom management.
ReFeReNCeS
1. Reinmller, B. (2001). People as a Contamination Source
- Clothing Systems. In: Dispersion and Risk Assessment of
Airborne Contaminants in Pharmaceutical Cleanrooms. Royal In-
stitute of Technology, Building Services Engineering, Bulletin
No. 56, Stockholm, pp. 54-77
2. Hyde, W. (1998). Origin of bacteria in the clean room and
their growth requirements. PDA J Sci Technol; 52:154164
3. Sandle, T. (2011). A Review of Cleanroom Microflora: Types,
Trends, and Patterns, PDA Journal of Pharmaceutical Science
and Technology, 65 (4): 392-403
4. Proksch E.; Brandner J.M.; Jensen J.M. (2008) The Skin: An
Indispensable Barrier, Exp. Dermatol. 17 (12): 106372
5. Whyte, W. (1981) Setting and impaction of particles into con-
tainers in manufacturing pharmacies, J. Paren. Sci. Technol.,
36: 255-268
6. Roth, R.R. and James, W.D. (1988): Microbial Ecology of the
Skin. Annu. Rev. Microbiol. Vol. 42, pp. 44164
7. Grice, E.A., Kong, H.H., Renaud, G., Young, A.C. (2008).
A diversity profile of the human skin microbiota. Genome
Research.18:1043-50. (PMID:18502944)
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8. Costello, E.K., Lauber, C. L., Hamady, M., Fierer, N., Gordon,
J.I., Knight, R. (2009). Bacterial community variation in
human body habitats across space and time, Science, 326:
16941697
9. Chen YE, Tsao H. (2013). The skin microbiome: current
perspectives and future challenges, J Am Acad Dermatol.
69(1):143-55
10. Grice, E.A., Kong, H.H., Conlan, S. et al (2009). Topographi-
cal and Temporal Diversity of the Human Skin Microbiome,
Science, 324: 1190 1192
11. Gao, Z., Tseng, C.H., Pei, Z., and Blaser, M.J. (2007). Molecu-
lar analysis of human forearm superficial skin bacterial biota.
Proc. Natl. Acad. Sci. 104: 29272932
12. Kong, H.H. and Segre, J.A. (2012). Skin Microbiome: Looking
Back to Move Forward, Journal of Investigative Dermatology,
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13. Findley, K., Oh, J. Yang, J., Conlan, S. et al (2013). Topo-
graphic diversity of fungal and bacterial communities in
human skin, Nature, 498(7454):367-70. doi:10.1038/na-
ture12171
14. Cogen A.L, Nizet, V. and Gallo, R.L. (2008): Skin Microbiota:
A Source of Disease or Defence?, Br. J. Dermatol., 158 (3): 442-
55
15. Ramstorp, M. (2011) Microbial Contamination Control in
Pharmaceutical Manufacturing. In Saghee, M.R., Sandle, T.
and Tidswell, E. (Eds.) Microbiology and Sterility Assurance in
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Delhi, pp615-701
16. Sandle, T. (2006) The use of polymeric flooring to reduce
contamination in a cleanroom changing area, European Jour-
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17. Kampf, G., Kramer, A. (2004). Epidemiologic Background of
Hand Hygiene and Evaluation of the Most Important Agents
for Scrubs and Rubs, Clinical Microbiology Review, p. 863893
18. Sutton. S., (2009). Hand Washing-A Critical Aspect of Per-
sonal Hygiene in Pharma, Journal of Validation Technology, 15
(4): 50-55
19. Spaudling, E.H. (1964) Alcohol as a surgical disinfectant,
AORN J., 2: 67-71
20. Klein, M. and DeForest, A. (1963) The inactivation of viruses
by germicides, Chem. Specialist Manufact. Assoc. proc., 49: 116-
118
21. Morton, H.E. (1950) The relationship of concentration and
germicidal efficiency of ethyl alcohol, Ann N.Y Acad. Sci, 50:
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22. Morton, H.E. (1983) Alcohols. In Block, S.S. (Ed.) Disinfection,
Sterilisation and Preservation, Philadelphia: Lea and Febiger,
pp225-239
 Xxxxxxx

PAT: Using PAT to

Support the Transition
from Cleaning Process
Validation to Continued
Cleaning Process
Verification | IVT
XXXXXX

ABSTRACT
In accordance with the 2011 FDA guidance for industry, Process Valida-
tion General Principles and Practices, there is a requirement to continue to
verify equipment cleaning processes. This continued verification would
reduce the risk of cross contamination, and consequently, the accept-
able residue limits could be increased to reflect this reduced risk. A
combination of visual residue limits and rapid analytical technologies
for the quantification of residues may lead to a reduced risk of cross
contamination for the patient while at the same time result in signifi-
cantly reduced manufacturing cycle times. This paper discusses the
current cleaning paradigm and the changes continued cleaning process
verification could bring for small molecule pharmaceutical manufactur-
ers. Technologies that offer potential for increased automation of clean-
ing validation are introduced.

INTRODUCTION
Cleaning can generally be defined as the removal of unwanted contami-
nants. The objective of a cleaning process is primarily to ensure safety,
efficacy, and quality of product subsequently manufactured in the same
equipment. Cleaning validation is the documented evidence demon-
strating the effectiveness of a cleaning procedure based on pre-deter-
mined acceptance criteria. It is performed to assure that production ma-
terials that come into contact with manufacturing equipment surfaces
are not contaminated or adulterated. A graphical representation of the
typical cleaning process development and validation process is detailed
in Figure 1. It summarizes the typical process and demonstrates the
extensive workload associated with cleaning process development and
validation.
Figures 2 and 3 summarize the current cleaning procedure develop-
ment and validation approach. This approach is time-consuming and
costly. It can significantly inhibit clinical trial manufacture activities
because of once-off swabbing requirements following each mini-batch
manufactured. It can also inhibit on-going commercial manufacturing
activities due to the burden associated with cleaning procedure valida-
tion and routine monitoring.
Within the FDA guidance document on process validation (1),
process validation is defined as the collection and evaluation of data,
from the process design stage throughout production, which establishes
scientific evidence that a process is capable of consistently delivering
quality products. Process validation involves a series of activities taking

Special edition: Cleaning Validation 39

Xxxxxxx
Figure 1: Typical cleaning process development and validation process.
Figure 2: Current cleaning procedure development process flow.
place over the lifecycle of the product and process.
The guidance describes the process validation activi-
ties in three stages: process design, process qualifica-
tion, and continued process verification.
While the greatest focus of this guidance docu-
mentation has been related to manufacturing process
validation, the same three stages of validation have
relevance to cleaning process validation. It is likely
that the demonstration of continued cleaning process
verification is expected by regulatory bodies in order
to demonstrate sufficient control of the cleaning
process.
With the increased frequency of verification associ-
ated with such a philosophy, it is logical that the level
40 Special edition: Cleaning Validation
Figure 3: Current cleaning procedure validation process flow.
of risk of cross-contamination of subsequent formu-
lations would reduce accordingly. As a result, the
acceptable residue limits (ARLs) currently associated
with cleaning validation may no longer be appropri-
ate. This is because the safety factors built into such
calculations may have been included as a result of
the snapshot-in-time nature of a cleaning validation
activity.
VISIBLE-RESIDUE LIMITS DURING CLEANING
PROCEDURE DEVELOPMENT AND VALIDATION
A visible-residue limit (VRL) is the quantity of target
residue (usually API) remaining on manufacturing
equipment surfaces when it has reached a visually de-
tectable level. An ARL is the amount of target residue
(usually API) that remains on manufacturing equip-
ment surfaces and carry over to the next formulation
with no pharmacological or adulteration risk. This is
typically calculated using 10ppm or 1/1000th mini-
mum daily dose as outlined in Figure 1.
With a shift to continued verification of a clean-
ing procedure following every clean and the reduc-
tion of cross-contamination risk, the associated
ARLs will increase, leading to greater numbers
of ARLs exceeding VRLs. This may subsequently
enable greater use of a VRL approach to continued
verification. The use of visual inspection as an as-
sessment criterion for equipment cleaning effec-
tiveness has always been a component of cleaning
programs. Mendenhall proposed the use of only
visual examination to assess equipment cleanliness
in 1989 (2). He summarised that visible cleanliness
criteria were more rigid than quantitative calcula-
tions and, therefore, adequate. FDA acknowledges
the use of visually clean criteria for product-dedi-
cated equipment. LeBlanc has also reviewed the use
of visual examination as the sole acceptance criteria

Table 1: Advantages and limitations of visible-residue limits (VRLs) during
cleaning procedure development and validation.
Figure 4: A process flow of proposed cleaning procedure development
and validation activities.
for cleaning validation (3).
Forsyth et al. (4, 5) propose that VRLs could be
adopted as the primary acceptance criteria during
cleaning evaluation activities if the VRL is calculated
to be less than the ARL. They cited the following
advantages and limitations detailed in the Table.
As is outlined in the Table, there are still vulnera-
bilities of such an approach, in particular the person-
to-person variability of visually clean equipment
and the variable lighting conditions associated with
commercial-scale equipment. Even with considerable
training and experience these risks may remain.
There is an opportunity to utilize rapid at-line and
on-line automated technologies to compensate for the
deficiencies within the VRL approach. A combination
of both approaches (i.e., VRLs and automated residue
detection systems) could support the pharmaceutical
manufacturing sector to realize a significantly robust
cleaning process aligned with reduced manufactur-
ing cycle times. This proposed paradigm is outlined
in Figure 4. Figure 4 provides an overview of the
proposed alternative approach to cleaning procedure
Xxxxxxx
development, process qualification, and continued
verification. Considerable quality assurance, time and
resource, and financial benefits may be realized from
adopting such an approach.
ALTERNATIVE TECHNOLOGIES FOR CONTINUED
VERIFICATION OF
Some examples of alternative technologies currently
employed or that show potential for continued verifi-
cation of equipment cleaning processes either as lab,
at-line, or in-line approaches are detailed as follows.
Lab-based Technologies
Lab-based technologies include ion-mobility spec-
trometry (IMS), total organic carbon (TOC), and
liquid chromatography-mass spectrometry-mass
spectrometry (LC-MS-MS).
Ion-mobility spectrometry. IMS is a separation
technique, similar to time-of-flight mass spectrom-
etry, that distinguishes ions of a given compound
based on their velocities through a drift tube under
the influence of a weak electric field. To perform the
IMS analysis, a sample solution containing a com-
pound of interest is injected by an autosampler onto a
polytetrafluoroethylene (PTFE) substrate and allowed
to dry. The sample is introduced by heating the
substrate to circa 290C, which results in desorption
or vaporization of the sample into an inlet tube (6).
Primary ion formation occurs through atmospheric
pressure chemical ionisation (APCI) using nickel-63
as the radioactive source. Following many collisions,
product ions are formed and gated into the drift tube
(6). These ions based on size, shape, and charge travel
through the drift tube toward the detector at dif-
ferent velocities. In contrast to mass spectrometers,
separation of ions is based on a size-charge relation-
ship rather than a mass-charge ration. It is a fast and
specific off-line tool for verifying the cleanliness of
pharmaceutical equipment. Recovery percentages and
standard deviations for IMS samples are consistent
with those obtained with HPLC analysis, but sample
throughput of IMS is about 50 times faster (6). The
disadvantages associated with such a method are that
it is generally an off-line analytical tool that requires
sample swabbing and preparation activities. In addi-
tion, significant method development time is required
prior to implementation. However online, real time
applications are becoming available.
Total organic carbon. TOC analysis is a method
that has been used successfully for monitoring water
quality, in particular the quality of water for injection
used in pharmaceutical manufacturing. It offers a
number of distinct advantages over other commonly
used residual testing methods. A typical method may
Special edition: Cleaning Validation 41
Xxxxxxx
involve acidification of a swab sample followed by
sparging with purified air to remove inorganic carbon
from solution as carbon dioxide gas. The organic
compounds remaining in solution are then oxidized
to carbon dioxide in, for example, a combustion
tube packed with a catalyst at 680C (7). The carbon
dioxide produced in this manner passes through a
dehumidifier and halogen scrubber before it reaches
a cell where it is quantified by a non-dispersive in-
frared detector tuned to the highly specific carbonyl
frequency in the 1-700cm-1 range (7). The signal
response is compared with a calibration curve gener-
ated by analysis of solutions of potassium biphthalate
corresponding to known levels of organic carbon.
The disadvantages associated with such a method are
that it is non-specific and an at-line analytical tool
that requires sample swabbing and preparation activi-
ties (7).
LC-MS-MS. For low-dose compounds, equipment
requiring low residue limits, and compounds lacking
strong chromophores, the sensitivity and selectivity
of LC-MS-MS facilitates rapid detection of low levels
of residues of active pharmaceutical ingredients (8).
Like IMS, the disadvantages associated with LC-
MS-MS are that it is an off-line analytical tool that
requires significant method development, sample
swabbing, and preparation activities.
At-line Technologies
ATP luminescence. Adenosine triphosphate (ATP)
luminescence-based technologies may be used to
evaluate the level of microbial contamination follow-
ing the completion of an equipment cleaning process.
ATP is a marker for cell viability because it is present
in all metabolically active cells where the concentra-
tion declines rapidly when the cells undergo necrosis
or apoptosis. The approach is based on the produc-
tion of light caused by the reaction of ATP with
added luciferase and D-luciferin (9-11). The emitted
light is proportional to the ATP concentration within
certain limits. The limitation associated with com-
mon luciferase assay technology is the short half-life
of the light emission. This flash-type signal requires
luminometers with reagent injectors to measure the
quick reaction. Prior to addition of luciferase, there
is a requirement to lyse the cells so that the ATP is
released.
This is a rapid method of swab analysis that is ac-
cepted within the food and beverage industries.
It may have application within sterile and aseptic
operations in particular. However, it is a non-specific
method and has the potential for contamination by
human somatic cells if sample-handling procedures
42 Special edition: Cleaning Validation
are not sufficiently rigorous (9-11).
0n-line Technologies
UV-Vis spectrophotometry. UV-Vis technologies
monitor rinse effluent to quantify the level of API
contained. UV-Vis spectroscopy is the measure-
ment of the wavelength and intensity of absorption
of near-ultraviolet and visible light by a sample.
Ultraviolet and visible light are energetic enough
to promote outer electrons to higher energy levels.
UV-Vis spectroscopy is usually applied to molecules
and inorganic ions or complexes in solution. The
UV-Vis spectra have broad features that are of lim-
ited use for sample identification but are useful for
quantitative measurements. The concentration of an
analyte in solution can be determined by measur-
ing the absorbance at some wavelength and applying
the Beer-Lambert Law. The light source is usually a
hydrogen or deuterium lamp for UV measurements
and a tungsten lamp for visible measurements. The
wavelengths of these continuous light sources are
selected with a wavelength separator such as a prism
or grating monochromator. Spectra are obtained by
scanning the wavelength separator and quantitative
measurements can be made from a spectrum or at a
single wavelength. When the level of API in the rinse
effluent reaches a pre-determined acceptable low
level, the cleaning process end-point is reached. This
is a novel approach that has significant benefits for
identification of cleaning process endpoint.
One disadvantage identified would be the challenges
of wavelength accuracy. Wavelength accuracy is de-
fined as the deviation of the wavelength reading at an
absorption band or emission band from the known
wavelength of the band. The wavelength deviation
can cause errors in the qualitative and quantitative
results of the UV-Vis measurement, thereby affecting
the accuracy and the sensitivity of the method (12). A
second disadvantage, similar to other rinse studies, is
that it may not detect API if it is adhered to the vessel
wall.
Single point near infrared (NIR). In situ infrared
reflection-absorption spectroscopy (IRRAS) has the
potential to provide a more rapid analysis than the
existing methods. Modern Fourier Transform-Infra-
red (FT-IR) instruments are quite stable and have
sufficient sensitivity. Infrared fibreoptics based on
chalcogenide glasses can allow sampling up to several
meters from the spectrometer (13-16). Multivariate
chemometric tools, such as partial least squares (PLS)
regression, allow implicit modelling of unquantified
interfering species and instrumental artifacts. For
cleaning validation, the physical presentation of the
contamination will depend on the solvent, the chemi-

cal nature and quantity of the residual material, and
the substrate surface. Mehta et al. (14), Hamilton et al.
(14), and Teelucksingh et al. (16) demonstrated that
IRRAS calibrations can be established for individual
surfactants and APIs on metallic and glass surfaces,
for an API in the presence of a surfactant on a glass
surface, and for an API in the presence of excipients
on a stainless steel surface (13-16). Single point NIR
has potential applications for cleaning verification
purposes. Its main advantage over traditional swab-
HPLC techniques is that it samples those contami-
nants that are present on the surface, rather than
those that can be removed by the swab coupled with
its speed of analysis. The primary limitation associ-
ated with the method is the restricted field of analysis
and whether it is sufficiently large and representative
to assess the success of the cleaning activity.
NIR-chemical imaging. Chemical imaging (CI) is an
emerging platform technology that integrates conven-
tional imaging and spectroscopy to attain both spatial
and spectral information from an object (17). It has
not to date been evaluated as a technology to quantify
residual levels of active ingredients and detergents
from equipment surfaces. However, based on its
broad applicability and sensitivity, it has potential for
cleaning validation. Near infrared-chemical imaging
(NIR-CI) is the fusion of near-infrared spectroscopy
and image analysis. It can be used to visualize the
spatial distribution of the chemical compounds in a
sample providing a chemical map of a region (Figure
5). Each sample measurement generates a hyper-
spectral data cube containing thousands of spectra.
An important part of a NIR-CI analysis is the data
Figure 5: Schematic representation of hyperspectral imaging hypercube
showing the relationship between spectral () and spatial (X, Y) dimen-
sions (17).
Xxxxxxx
processing of the hyperspectral data cube (18).
The large number of individual spectra acquired
across the spatial dimension of heterogeneous com-
pounds provides a basis from which relative concen-
trations can be determined for each spatial location.
Alternatively, these individual concentrations may
be added together to give the total concentration of a
specific material within the sample area.
Chemical images are made up of hundreds of
contiguous wavebands for each spatial position of a
target studied. Consequently each pixel in a chemical
image contains the spectrum of that specific posi-
tion. The resulting spectrum acts like a fingerprint
that can be used to characterize the composition of
that particular pixel. There are two basic methods to
construct the chemical image. One method involves
acquisition of simultaneous spectral positions. The
object is moved underneath an imaging spectro-
graphthis is termed pushbroom acquisition. The
other method involves keeping the image field of
view fixed and obtaining images one wavelength after
anotherthis is termed staring imager configura-
tion (17). This method is of particular interest within
the pharmaceutical industry for sample analysis and
would be considered to pose the greatest potential for
use as a cleaning verification device. This is primar-
ily because the sample can remain stationary and the
field of view would be comparable with that currently
used during conventional swabbing techniques (circa
25cm2).
Point-source spectroscopic assessments do not
provide information on spatial distribution of dif-
ferent constituents. In other words, NIR and Raman
spectroscopy can only provide information on a very
narrow sample site. NIR-CI can provide informa-
tion on a substantial sample site thereby making it
applicable to analysis of equipment surfaces dur-
ing cleaning validation. Challenges with regards to
method sensitivity for such residual levels will need
to be overcome before this technology becomes com-
mercially viable.
CONCLUSIONS
Rapid technologies would remove some significant
variables identified with VRLs and enable an analyti-
cal technique that could significantly reduce the time
and cost associated with the cleaning procedure.
They could also support the transition away from
once-off cleaning validation towards continued clean-
ing process verification. This would be in-line with
regulatory expectations and pharmaceutical manu-
facturing needs.
The increased frequency of equipment cleaning
process verification will reduce the risk of active and
detergent cross-contamination and thereby enable
higher acceptance criteria for active and detergent
Special edition: Cleaning Validation 43
Xxxxxxx

carryover. As the pharmaceutical manufacturing 12. Lam, Herman, Performance of UV-Vis Spectrophotometers,
industry transitions towards these continued verifica- Laboratory FocusGazette Edition, Issue, 8, April 2000.
tion philosophies, the requirement for rapid mobile 13. Hamilton et al., Grazing-angle fiber-optic IR reflection-ab-
analytical technologies has become essential in order sorption spectrometry (IRRAS) for in situ cleaning valida-
to sustain robust and lean equipment cleaning pro- tion, Org. Process Res. & Devel., 9(3), 337-343, 2005.
cesses. 14. Mehta, N.K.; Goenaga-Polo, J.; Hernandez-Rivera, S.P.; Her-
nandez, D.; Thomson, M.A.; Melling, P.J. Biopharmacology, 15,
REFERENCES 36-71, 2002.
1. FDA, Guidance for Industry, Process Validation General Prin- 15. Hamilton, M.L.; Persoton, B.B.; Harland, P.W.; Williamson,
ciples and Practices, Current Good Manufacturing Practices, B.E.; Thomson, M.A.; Melling, P.J.; Org Process Res. Dev., 9,
January 2011. 337-343, 2005.
2. D.W. Mendenhall, Cleaning Validation, Drug Development 16. Teelucksingh, N.; Reddy, K.B., Spectroscopy, 20, 16-20, 2005.
and Industrial Pharmacy 15 (13), 2105 2114, 1989. 17. Gowen.A, ODonnell.C.P, Cullen.P.J, Bell.S.E.J, Recent appli-
3. D.A. Le Blanc, Visually Clean as a Sole Acceptance Criteria cations of Chemical Imaging to pharmaceutical process moni-
for Cleaning Validation Protocols, PDA J. Pharm Sci. And Tech- toring and quality control, European Journal of Pharmaceutics
nology, 56 (1), 31-36, 2002. and Biopharmaceutics 69, 1022, 2008.
4. Richard J. Forsyth et al., Risk-Management Assessment of 18. Ravn,C. Skibsted,E. Bro, R. Near-infrared chemical Imaging
Visible-Residue Limits in Cleaning Validation, Pharm. Tech- (NIR-CI) on pharmaceutical solid dosage forms-Comparing
nol. 30, 104114 September 2006. Common calibration approaches, Journal of Pharmaceutical
5. Richard J. Forsyth, Ruggedness of Visible Residue Limits for and Biomedical Analysis 48 554561, 2008. JVT
Cleaning Validation, Pharm. Technol. 33, 102 111 March
2009. ARTICLE ACRONYM LISTING
6. Elizabeth Galella et al., Cleaning Verification: Method De- APCI Atmospheric Pressure Chemical Ionisation
velopment and Validation using Ion Mobility Spectrometry, API Active Pharmaceutical Ingredient
Pharm. Technol. 33, 60 63 July 2009. ARLs Acceptable Residue Limits
7. A. Strege, Terry L Stinger, Brett T. Farell and Avinash L Lagu, ATP Adenosine triphosphate
Biopharm International, April 1996. CI Chemical Imaging
8. Kevin J. Kolodsick et al., Enhancing Drug Develoment by FT-IR Fourier Transform-Infrared
Applying LC-MS-MS for Cleaning Validation in Manufactur- IMS Ion-Mobility Spectrometry
ing Equipment, Pharm. Technol. 30, 56 71 February 2006. IRRAS Infrared Reflection-Absorption Spectroscopy
9. Kangas L., Grnroos M. and Nieminen A.L., Biolumines- LC-MS-MS Liquid Chromatography-Mass
cence of cellular ATP: a new method for evaluating agents in Spectrometry-Mass Spectrometry
vitro, Medical Biology, 62,338 343, 1984. NIR Near Infrared
10. Lundin A., Hasenson M., Persson J. and Pousette A., Estima- NIR-CI Near Infrared-Chemical Imaging
tion of biomass in growing cell lines by ATP assay, Methods PTFE Polytetrafluoroethylene
Enzymol. 133, 27 42, 1986. TOC Total Organic Carbon
11. Crouch S.P.M., Kozlowski R., Slater K.J. and Fletcher J., The UV Ultraviolet
use of ATP bioluminescence as a measure of cell proliferation VRL Visible Residue Limit
and cytotoxicity, J. Immunol. Methods, 160, 81 88, 1993.

44 Special edition: Cleaning Validation

 Keith Bader & Kelly Scalva

Translating Laboratory-
Developed Visual Residue
Limits to Process Area
Applications | IVT
Keith Bader and Kelly Scalva

ABStrAct
While considerable attention has been given to the development of
visual residue limits (VRLs) in a laboratory setting, translating the
bench scale values to the assessment of process surfaces has not yet
been thoroughly assessed. However, knowledge of both the critical
parameters that impact the determination of VRLs and the influence of
those parameters on visual inspection can provide a framework for the
development of a robust visual inspection program. Development of
such a program first entails the determination of constraints imposed
by equipment geometries and facility lighting. VRLs can then be deter-
mined for post-productions residues of concern, which, of course, car-
ries its own specific challenges. Once VRLs have been determined, they
cannot be immediately applied without considering certain strategic
cleaning program approaches and potential sources of variability.
Many factors influence how visual inspection will be conducted in a
manufacturing facility. Among the most critical are inspection condi-
tions in the facility, the condition of existing equipment surfaces, and
the physical characteristics of post-production residues deposited on
product contact surfaces. While many in industry embrace the im-
portance of visible residue limits (VRLs), few have a clear pathway to
translate VRLs determined in the laboratory to the manufacturing floor.
The intent of this paper is to provide the background and informa-
tion that will allow industry to formulate a plan of attack to practically
integrate VRLs into the visual inspection program. To begin, the best
starting place is to determine the constraints imposed by the manufac-
turing floor.

EquipmEnt inSpEction conditionS

Conducting a visual residue limit (VRL) determination study in the
laboratory must be completed such that important process equipment
parameters from the manufacturing floor are captured. To wit, the first
step is characterization of the conditions on the manufacturing floor
where visual inspection is put into practice. For any residue, there are
three parameters that must be considered for proper translation and
laboratory analysis: viewing angle, distance, and light intensity at the
product contact surface. The most critical variable for visual inspection
is, of course, the room lighting, so it is important to understand and
characterize lighting in the facility to properly later simulate it at the
bench scale.
The interior task lighting generally conforms to standards set by a
professional organization such as the Illuminating Engineering Society
of North America (IESNA). In the past, over-lighting was the norm
with standard luminance regularly reaching 1200 lux (1). Lighting was

Special edition: Cleaning Validation 45

Keith Bader & Kelly Scalva
figure 1: Determination of Viewing Distance and Angles.
also primarily done with less efficient incandescent
and full-spectrum lamps that inherently provided
good color rendering. However, the advent of current
sustainable design, legislated energy conservation
requirements, and project engineers that target seem-
ingly non-critical lighting designs in value engineer-
ing exercises means that manufacturers can no longer
assume that room lighting conditions will be consis-
tent or even adequate for visual inspection. Specifi-
cally, current legislation limits energy use for lighting
to 1.12.0 w/ft, depending on the space function,
forcing less experienced lighting designers to select
options that may not provide equivalent performance
to historical lighting choices.
Indeed, Forsyth et al noted that lighting levels can
vary from 400 to 1200 lux in a facility; such a range
is representative of that found in most manufactur-
ing facilities (2). Such variability is often unavoidable
as facility and process layout can change over time
to meet manufacturing needs, leading to shadows or
reflections unaccounted for in the original design.
Accordingly, it is recommended that the lighting be
assessed and recorded through the use of commonly
available light meters to acquire custom-tailored
ranges for developmental VRL studies.
The most convenient way of recording these
numbers is to record them on a layout drawing of the
facility. Furthermore, it is useful to note the style of
46 Special edition: Cleaning Validation
light fixtures and whether the color temperature of
the lamp closely replicates sunlight. Lighting at the
product contact surfaces within process vessels is a
unique and involved set of measurements to acquire,
as the distances and angles from which operators
view the product contact surface is dependent upon
available viewing ports and the differences in tank
configuration, shown in Figure 1. Measurement of
the distances and angles is most reliably performed in
the field using a laser distance meter and clinometers;
however, this information can also be determined
from mechanical drawings of the equipment.
As process equipment is surveyed, it is advisable
to develop a baseline record of known blemishes,
marks, imperfections, and visual obstructions to
ensure that they can be distinguished from post-pro-
duction residues. Establishing this baseline for each
process tank and training inspectors on the location
and appearance of these fingerprint imperfections
are critical to prevent the development of a viewing
bias that may impact the care used when inspecting
process equipment surfaces.
Characterized and appropriately restricted view-
ing angles developed during the survey will allow for
better translation of the VRL from the laboratory to
the floor.
Conditions in areas where manually cleaned
equipment or small parts are inspected after they
are cleaned out of place are important in that many
pieces of manufacturing equipment used for closed
processing operations do not allow for reliance on
ambient lighting and require the specification of an
independent light source for visual inspection. Ac-
cordingly, it is important to determine the maximum
inspection distance based on the size and configura-
tion of vessels and other closed processing equip-
ment. Once this is determined, it can be used not
only as a selection criterion for a light source but also
as one of the constraints for conducting laboratory
VRL studies.
thE SElEction of A light SourcE for ViSuAl
inSpEction
Before conducting the laboratory study or send-
ing technicians into the field to inspect equipment,
it is important to specify a hand-held light source.
Specifying a hand-held or tank-mounted light source
carries some additional considerations. This requires
understanding some lighting design terminology and
heuristics. The hand-held light should be selected
with three primary characteristics taken into con-
sideration: color temperature, color rendering index
(CRI), and light output or luminous flux.
Color temperature, or chromaticity, refers to the
color of the light emitted from the light source. Colors
similar to the warm yellow orange of a candle flame

Source CRI Chromaticity (K)
Incandescent 100 2800K
Halogen 100 2900K
Fluorescent 6290 27006000K
Mercury 15 5700K
High Pressure Sodium 22 2000K
Metal Halide 6570 27004000K
Pulse Start Metal Halide 6575 30004000K
are assigned a temperature of 1800 Kelvin; up the
scale is the bluish light cast by metal halide and the
fluorescent light sources that are assigned a color
temperature of 4200 Kelvin (3). The ability of observ-
ers to accurately perceive colors is also somewhat de-
pendent on the chromaticity of the light source based
on the second characteristic noted above, CRI.
CRI is quantified on a scale that assigns the light
source a rating from 0 to 100, with the best rendition
of color achieved with a CRI of 100. For detail-orient-
ed physical inspection work and accurate evaluation
of residues on process equipment surfaces, the CRI
of a light source should, at a minimum, be at a value
of 65 or greater (4). The general relationship between
color temperature, CRI, and various light sources is
shown in the Table.
Finally, the light output of the source should be
considered. Many are also tempted to get the bright-
est light they can find; however, this can lead to
problems with detecting residues because excessive
glare can actually inhibit detection. The inspection
of process vessels is thus facilitated by selecting a
tank light or handheld light source that replicates
levels of lighting equivalent to the recommended for
ambient task lighting within the vessel. Most light
manufacturers, however, do not provide the luminous
intensity at various distances from the source. Hand-
held light output, when rated, is often quantified
using lumens out-of-the-front (OTF). On occasion, a
specific intensity may be provided in Lux (lumen/m 2)
for some lamps at a specific distance.
To convert intensity at a given distance to one
matching the distances determined for the produc-
tion equipment in the field, it is important to know
that light intensity follows an inverse square relation-
ship relative to distance, as shown in Figure 2. As
the distance from the light source increases, the light
must diffuse over a larger surface thereby reducing
the surface brightness in accordance with a one over
d squared relationship. Alternatively, an inspec-
tion light of appropriate intensity can be determined
empirically in the laboratory by using a light meter
at the appropriate distance. For the most part, an ac-
ceptable CRI and chromaticity are provided in hand-
held light sources by either incandescent or halogen
Keith Bader & Kelly Scalva
figure 2: Light Intensity vs. Distance.
lamps as shown in the Table. Furthermore, a flash-
light with an adjustable spot size can also be used to
vary the intensity and provide greater flexibility over
a range of distances.
lABorAtory Vrl dEtErminAtion StudiES
Translating the angle, distance, and lighting param-
eters from the onsite equipment to the confines of
the laboratory is best examined within a range of
values. Most commonly tested in the lab are three
angles (30, 45, 90), two to three distances (clos-
est and farthest away), a minimum of two different
lighting intensities (standard room task lighting and
the intensity from a hand-held light source), and a
variety of materials of construction (MOC). Assum-
ing the viewing variables were tightly and accurately
measured at the manufacturing site, the only other
test variables to be investigated in the laboratory
are the process soils (in varying concentrations),
materials of construction, and the assessment opera-
tors. As the potential variability in technicians is
the most difficult parameter to control and replicate,
they are discussed in greater detail in a subsequent
section.
ExpErimEntAl dEtAilS And logiSticS
First, coupons composed of materials of construc-
tion representative of process equipment should be
obtained (see Figure 3) and cleaned thoroughly to
remove any possible residue on the coupons; such
as dust, mechanical cutting oils; or, if the coupons
are not new, residue from previous tests. Typically,
a cleaning regimen consistent with United States
Pharmacopeia (USP) <1051> for cleaning glassware
employed for total organic carbon analysis will effec-
tively remove materials that can potentially confound
results. Cleanliness can then be confirmed using the
American Society for Testing and Materials (ASTM)
water break free test (5, 6). Clean coupons are then
rigorously inspected for visual cleanliness, surface
imperfections, water spots, discoloration, or oxidation
before use in the VRL Study. Coupons that appear to
have any noticeable imperfections are rejected from
the study.
Special edition: Cleaning Validation 47
Keith Bader & Kelly Scalva
figure 3: Coupons of Representative MOCs
Following a visual inspection of the coupons to
ensure their cleanliness, the process soils of inter-
est are diluted into a range of concentrations. These
concentration ranges are then spiked with equiva-
lent volumes onto the center of the coupon surface
and allowed to dry. Coupons commonly are dried at
ambient conditions overnight to ensure dryness, but
they can be dried at elevated temperatures depending
on process conditions. A visual target range is then
determined to find the appropriate loading density
and loading solvent for use in VRL testing.
Purified water is the common solvent used to make
the residue dilutions; however, surface tension ef-
fects combined with some process soils can create a
very visible ring around the periphery of the original
spiking area. As water dries at the edges of the spiked
solutions, it concentrates the residue, making them
more visible and quantification of the mass per unit
surface area more difficult. This concentrates the
residue to a greater degree in the laboratory setting
than that found on process equipment. Thus, it is
sometimes nessary to create a dilution mixture that
48 Special edition: Cleaning Validation
figure 4: Determining the Visibility Inflection Point.
includes a mixture of residue, purified water, and
isopropyl alcohol (IPA) to reduce the solution surface
tension and more uniformly distribute the residue
over the spiking area as the liquids dry, thereby
preventing concentration around the outer perimeter
or center of the spiking area. The exact ratios of IPA
to water must be determined on a per soil basis as
well as the solubility because each residue is differ-
ent. Furthermore, chemical interactions or protein
denaturation that alters the visual characteristics of
the soil should also be considered.
Once drying effects have been effectively contend-
ed with, the threshold concentration of each process
soil must be determined before multiple technicians
are screened. Specifically, the approximate concentra-
tion range visibility inflection point, when a residue
goes from visible to not visible, can be determined, as
shown in Figure 4.
Creating the initial spiking solutions in order of
magnitude intervals works well to initially narrow
the range. For example, a serial dilution of 1 ml resi-
due solution in 10 ml solvent followed by 1 mL of the
second solution in 10 mL of water, and so on, allows
a simple way to determine the range of concentra-
tions to be used for the VRL intermediate precision
testing assessment.
To prepare the coupons for testing, a consistent
volume of spiking solution is then spiked onto cou-
pons over a reproducible surface area. The concentra-
tions applied to the coupons span the range of Vis-
ible, Slightly Visible, and Not Visible as determined
in the initial bulk VRL screening exercise described
above. At least three replicates of each concentration
per material of construction should be used to ensure
statistical significance within the results. Four opera-
tors with 20/20 or corrected 20/20 vision are then
selected to establish the VRL intermediate precision
testing at different viewing angles and distances un-
der ambient and additional hand-held lighting.
To better simulate visual inspection conditions
on the manufacturing floor, it is important that the
coupons are placed on a background made of visually
similar material. The visually similar material reduc-
es operator bias during the laboratory study so that
contrast from the viewing background will not be
an issue. Thus, when testing the viewing of different
materials of construction, the background for these
assessments must either be constructed of the same
material as the coupon or must be consistent with the

figure 5: VRL Determination Conditions Based on Manufacturing Area
Assessment.
appropriate viewing parameters employed for the pro-
cess equipment. For example, if the major material of
construction is 316L SS EP20Ra with minor materi-
als such as borosilicate glass (commonly site glasses)
and/or PTFE (primarily tested to simulate valves), the
minor materials should have viewing backgrounds
equivalent or comparable to the major material of
construction. For example, 316L, 316, and 304 stain-
less steel would be visually comparable materials. If
glass were the major material of construction, match-
ing the background could also be somewhat complex
since interactions between process equipments and
the manufacturing area floors or walls could lead to a
more difficult situation to emulate in the laboratory.
Other variables to consider are the impact of personal
protective equipment (PPE) worn in the manufactur-
ing space, such as goggles, safety glasses, laboratory
coats, coveralls, or scrubs. The spiked coupons are
then arranged in a viewing area with controlled light-
ing and background.
To control for variations in visual acuity, operators
were screened to ensure that they had either correct-
ed or uncorrected 20/20 vision. Those without 20/20
vision were excluded from the study. Screening was
conducted by first asking each operator if they had
had a professional eye exam within the last twelve
months. If so, the operator was asked to view two dif-
ferent Snellen eye charts from distances of 6 ft. and
10 ft., respectively. A passing result for this portion
of the test required that the operator could correctly
recite the line on the chart corresponding to 20/20
without reading any of the letters incorrectly. This
practice is recommended to qualify technicians both
in the laboratory and on the manufacturing floor.
When transferring the measured angles and distances
to the laboratory, keep in mind that technicians
Keith Bader & Kelly Scalva
in the lab will not have the geometric restrictions
imposed by ancillary equipment, viewing ports, and
manways, thereby necessitating artificial limitations
on the amount of movement they may instinctively
employ as they try to observe spotted coupons. A
representative arrangement of coupons is shown in
Figure 5.
rEcommEndAtionS for tESting
In any quality system, the integration of quality-
by-design principles starts with the education of all
personnel involved in the production of a therapeutic
substance. Specifically, when developing training ma-
terials for visual inspection of the process equipment,
operators should be informed of the importance of
the activity as an important orthogonal method for
the prevention cross contamination. This will help
prevent technicians from treating the activity as just
another task and rushing to complete it at the poten-
tial peril of patients receiving a particular therapeutic
compound.
In the laboratory setting, the study was most influ-
enced by the level of training of the visual assessment
operators. For example, operators who had experi-
ence cleaning coupons in the lab were much more
adept at dismissing imperfections on the surface of
coupons that may have otherwise confused those
who had never before conducted visual inspection or
VRL testing with residues. Not only is it important
for operators to know and understand why they are
completing a task, but it is just as important for the
operators to have the chance to study a dirty vessel
directly after a process run and at the end of the dirty
hold time in addition to having historic areas of con-
cern and vessel imperfections pointed out to them.
A well-educated operator trained to recognize the
appearance of process equipment at both its worst
and best relative to cleanliness is much more likely
to properly ascertain the presence of residues on
product contact areas. Specifically, operators should
also have the opportunity during training to visually
inspect the vessel directly after the cleaning cycle.
If possible, they should also be able to observe the
equipment in a clean, wet state and a completely dry
state since residue is typically better seen on dry pro-
cess surfaces. Knowledge of how the vessel appears
when the surfaces are covered in process residues
typically makes the cleanliness easier to assess if an
idea of the range of cleanliness is known.
Bias can also be avoided in the laboratory evalu-
ation by arranging the coupons both in a random
loading concentration order and in rows of, or greater
than, five coupons to prevent operators from memo-
rizing which coupons they identified as soiled under
a previous lighting, distance, and viewing angle
combination. Based on the authors experience, more
Special edition: Cleaning Validation 49
Keith Bader & Kelly Scalva
consistent results are also obtained if the operators
verbally relay whether the residue is visible rather
than be required to fill out protocol worksheets.
Overall, it was found that slight movement of the
flashlight was necessary for the operators to attempt
to see the residues; directing the light towards the
coupon led to excessive glare that hindered the ability
of the operator to view residue. Residues were typi-
cally noted when they were on the periphery of the
hand-held light source spot. It was also noted that
operators who viewed the surfaces longer generally
saw more residue than operators who quickly glanced
at the coupons. However, with the use of the flash-
light, operator-viewing duration increased by about
50%. Interestingly enough, visibility and detection
of dirty coupons decreased when operators used the
hand-held light over diffuse ambient lighting.
While the procedures described above may seem
fairly complete and extensive, there are still many re-
lated topical permutations warranting further inves-
tigation. For example, most studies, to date, focus on
the intact API; however, cleaning processes employed
for biopharmaceuticals often employ alkaline clean-
50 Special edition: Cleaning Validation
ing agents that degrade post-production residues. As
with any chemical change to a compound, the physi-
cal properties can change, including visibility of the
compound on process surfaces. Future studies will
investigate this and other similar considerations.
rEfErEncES
1. E. Teicholz, LIGHTING in Facility Design and Management
Handbook, (McGraw-Hill Professional, 2001), AccessEngineer-
ing.
2. R.J. Forsyth, V. Van Nostrand, and G. Martin, Visible-Resi-
due Limit for Cleaning Validation and its Potential Applica-
tion in a Pharmaceutical Research Facility, Pharmaceutical
Technology 28 (10), 5872, 2004.
3. T. Croft, W.I. Summers, F.P. Hartwell, PRINCIPLES AND
UNITS in American Electricians Handbook, Fifteenth Edition,
(McGraw-Hill Professional, 15th ed., 2009 2002 1996 1992
1987 1981 1970 1961).
3. R.C. Rosaler, Lighting in Standard Handbook of Plant Engi-
neering, Third Edition (McGraw-Hill Professional, 3rd ed.,
2002 1995 1983)
4. USP29NF24 <1051>, Cleaning Glass Apparatus, 2896
 Rizwan Sharnez

Cleaning Validation of
Multiproduct Equipment
Acceptance Limits for
Inactivated Product
Part IIApplication of the Comparable
Quality Approach to Intrasite Assessments

Rizwan Sharnez, Elizabeth Aisenbrey, Joel Bercu, David Binkley, and Arun Tholudur

New Perspectives on Cleaning is an ongoing series of articles dedicated

to cleaning-process development, validation, and monitoring. This column
addresses scientific principles, strategies, and approaches associated with
cleaning that are relevant in everyday work situations.
Reader questions, comments, and suggestions are requested for future
discussion topics. These can be submitted to Rizwan Sharnez at rsharnez@
amgen.com.

SUMMARY
For multiproduct cleaning validation, acceptable carryover of the previ-
ously manufactured active pharmaceutical ingredient (API) (Product A) into
the subsequently manufactured API (Product B) is determined through a
maximum allowable carryover (MAC) calculation (1). A limitation of the
conventional MAC approach is that if the previously manufactured API
becomes therapeutically inactive during cleaning, then there is limited value
in verifying removal of the API. Instead, it is more appropriate to demon-
strate that the carryover of the inactivated product between lots of different
products (i.e., A B, or B A) is acceptable.
If the API is inactivated when exposed to worst-case cleaning condi-
tions (i.e., conditions that are least conducive for inactivation), the compa-
rable quality (CQ) approach can be used to set acceptance limits for car-
ryover of the inactivated product (IP) (2). The CQ approach is designed to
ensure that the amount of inactivated Product A in the largest dose (LD)
of Product B (M LDIP AB ) is less than or equal to the amount of inactivated
Product A in the largest dose of Product A (M LD IP AA),

M LD
IP AB M LD
IP AA [Equation 1]

To apply the CQ criterion to cleaning validation, the above inequality

must be expressed in terms of measureable equipment and product pa-
rameters. An approach for expressing this criterion in terms of analytical
data (e.g., total organic carbon [TOC] swab or rinse data), product contact
surface area, and batch and dose sizes is described in this paper. The
resulting expression for the CQ criterion for intrasite assessments (i.e.,
where Product A is being manufactured at the same site where Product
B is being manufactured) is given by Equation 2. When this inequality is
satisfied, the amount of inactivated Product A in the largest dose of Prod-
uct B will be limited to the amount of inactivated Product A in the largest
dose of Product A.

Special edition: Cleaning Validation 51

Rizwan Sharnez
If the characterization data indicate that the previ-
ously manufactured API (i.e., API in Product A) is
inactivated when exposed to cleaning conditions, and
the CQ criterion is met, then the acceptance limits
for the process residue can be set based on the lowest
of the following two limits: process capability and 10
ppm limit for carryover of process residue.
A flowchart summarizing the methodology de-
scribed in this paper is shown in Figure 1. The CQ cri-
terion is derived in Appendix I and a worked example
is given in Appendix II.
The CQ approach was described in Part I of this
series. In Part II, the CQ approach is applied to intra-
site assessments; application of the CQ approach to
intersite transfers is described in Part III.
INTRODUCTION
Biopharmaceutical cleaning cycles are generally designed
to expose product contact equipment to extremes of
pH (pH 213) and temperature (60-80C) for several
minutes. The equipment may also be steam sterilized or
sanitized. Under these conditions, monoclonal antibod-
ies, therapeutic proteins, and other biological APIs are
known to degrade and denature rapidly, and become
therapeutically inactive (2-4).
Inactivation Studies
Inactivation of the API during cleaning can be assessed
by exposing the process soil to simulated cleaning
conditions at bench scale. The inactivation studies are
designed to simulate full-scale cleaning conditions that
are least conducive (i.e., worst case) for inactivation (e.g.,
lowest ratio of cleaning solution to protein and shortest
exposure). The objective of these studies is to ascertain
whether the API in the process sample is inactivated
when exposed to worst-case cleaning conditions.
After exposure to the worst-case cleaning condi-
tions, the samples are titrated to a neutral pH and
cooled to 4C to minimize further degradation and in-
activation. Appropriate untreated controls are included
to assess the impact of any experimental artifacts and
potential matrix effects. The samples are then sub-
jected to SDS-PAGE and bioassay to determine the
degree of degradation and inactivation, respectively.
The samples may also be subjected to TOC analysis
to determine whether the inactivated product can be
adequately recovered by TOC.
The results of the small-scale studies can justifi-
ably be extrapolated to the full-scale cleaning process,
because degradation and inactivation are essentially
scale-independent phenomena (i.e., they depend on
cleaning parameters that are effectively independent
of spatial coordinates, namely time, temperature, con-
centration, and the ratio of cleaning solution to process
soil). It should be noted that degradation and inactiva-
tion studies are typically not performed at full scale
52 Special edition: Cleaning Validation
because the concentration of the degradants in clean-
in-place (CIP) samples is generally well below the limit
of detection (LOD) of SDS-PAGE and most bioassays.
In the small-scale studies, this issue is addressed by
using a soil to solution ratio (R) that is high enough
to ensure that the concentration of degraded product
in the sample is above the LOD of the assay. Note
that a higher R represents a milder condition from the
standpoint of degradation (i.e., higher R implies that a
smaller fraction of the total soil would degrade).
Implications for Cleaning Validation
The inactivation of the API during cleaning has impor-
tant implications for cleaning validation of multiproduct
equipment. Demonstrating that the product is inactivat-
ed during cleaning obviates the need to set limits based
on a conventional MAC assessment for the API (5). It also
eliminates the need to develop product specific assays for
cleaning validation (6).
The Comparable Quality Approach
If the API is inactivated when exposed to worst-case
cleaning conditions, the CQ approach can be used to
set acceptance limits for cleaning (2). The CQ approach
ensures that the amount of inactivated Product A in
the largest dose of Product B is limited to the amount
of inactivated Product A in the largest dose of Product
A. Thus, the CQ approach provides assurance that in a
single exposure the amount of inactivated Product A that
a patient taking Product B receives is less than or equal
to the amount of inactivated Product A that a patient tak-
ing Product A receives. The rationale for this approach is
that if one set of patients (i.e., those taking Product A) are
being exposed to a certain amount of inactivated Product
A, then it is acceptable for another set of patients (i.e.,
those taking Product B) to be exposed to a lesser or equal
amount of inactivated Product A.
The CQ approach is based on the premise that
an appropriate product quality assessment has been
completed on Product A to show that Product A is of
acceptable quality.
Differences in Patient Subpopulations and Frequency
or Duration of Dosing
For pharmacologically-active substances, therapeutic ef-
fects and toxicity that occur from a single peak exposure
or continually over time are important considerations for
setting acceptable limits for exposure. In order to study
the effects of key variables such as dosing frequency,
length of exposure, and sensitivity, active substances are
extensively tested in the patient subpopulations tak-
ing the drug. While these endpoints are important for
the study of active substances, they are not relevant to
degradants that are pharmacologically inactive. Instead,
the comparable CQ approach is more appropriate for
establishing limits for inactive degradants.
 Rizwan Sharnez

Perform
inactivation
study a

Is API in process sample

inactivated after exposure
to cleaning conditions? b
Yes No

If CQ criterion is Use conventional MAC

satisfiedc set acceptance approach to
limit (AL) for inactivated set acceptance limit (AL)
product (IP) d per Eqn. 2 for API e

Can IP be Can API be

recovered by recovered by
TOC? TOC?
Yes No Yes No

Is AL LOQ No Is AL LOQ No
of TOC? of TOC?

Use TOC to Develop Use TOC to Develop

demonstrate alternative assay demonstrate alternative assay
removal of to demonstrate removal of to demonstrate
IP removal of IP API removal of API
a
Inputs: process sample, reference standard, cleaning cycle, and equipment parameters
b
Based on bioassay analysis
c
If CQ criterion is not satisfied, the approach described in this paper is not applicable
d
Inputs: dose and batch sizes of product, surface area of dedicated and shared equipment,
and surface concentration of IP (refer to Appendix 1.2)
e
Inputs: dose and batch sizes of product, surface area and rinse parameters of shared equipments [8]

Figure 1: Flowchart for setting acceptance limits for multiproduct cleaning validation.

Effect of Cleaning Process
Note that for a given cleaning process for Product A,
the molecular weight distribution (MWD) of Product A
that is carried over into a subsequent lot of Product A
(intracampaign cleaning) will be similar to the MWD
of Product A that is carried over into a subsequent lot of
Product B (intercampaign cleaning). It is also important
to note that no additional inactivated product is intro-
duced into either product as a result of implementing the
CQ approach.
A literature search did not uncover methods for cal-
culating cleaning validation acceptance limits for the
carryover of inactivated product.
Setting Acceptance Limits
Based on Protein Inactivation
A methodology for setting acceptance limits for multi-
product equipment is summarized in Figure 1. Degrada-
tion and inactivation studies are first performed under
simulated worst-case cleaning conditions. If the sample
is shown to have no detectable activity (based on the
results of the bioassay), then the CQ approach is used
to set acceptance limits (AL) for the inactivated product
(left side of Figure 1). However, if the sample is shown to
have biologically active product, the conventional MAC
approach is used to set the AL for the previously manu-
factured product (APIA) (right side of Figure 1). If the
process residue (inactivated product or API, as the case
may be) can be recovered by TOC and the correspond-
ing AL the LOQ of TOC, TOC may be used to verify
removal of the process residue at full scale; otherwise, an
alternative assay may need to be developed.
COMPARABLE QUALITY CRITERION IN TERMS OF
MEASURABLE PARAMETERS
If the product becomes therapeutically inactive during
cleaning, then the acceptance limit for the inactivated
degradants of the previously manufactured product
(Product A) in the subsequently manufactured product
(Product B) can be set based on the CQ criterion. In
terms of the carryover of inactivated product, the CQ
criterion can be stated as follows.

Special edition: Cleaning Validation 53

Rizwan Sharnez
The amount of inactivated
LD
Product A in the largest
dose (LD) of Product B (M IP AB ) should be less than
or equal to the amount of inactivated
LD
Product A in the
largest dose of Product A (M IP AA) (2). Thus,
M LD
IP AB M LD
IP AA [Equation 1]
To apply this criterion to set acceptance limits for
inactivated product, the above inequality must be
expressed in terms of measureable equipment and
product parameters. An equation for expressing this
criterion in terms of analytical data, product contact
surface area, and batch and dose sizes is derived in
Appendix I. For intrasite assessments, where the
intracampaign cleaning cycle (AA) is similar to the
intercampaign cleaning cycle (AB), the result is as
follows (Equation AI-5):
Ni=1 [ CAA,i SA AA,i] SBS A LD B
[Equation 2]
Mj=1 [CAB,j SA AB,j ] SBS B LD A
where,
SAAA,i is the total product-contact surface area
for the ith system (CIP circuit) of the equipment
train used to manufacture Product A
N is the total number of systems that are used to
manufacture Product A
SAAB,j is the product-contact surface area
for the jth system of the equipment train that is
shared between Products A and B
M is the number of systems that are shared
between the two products
CAA,i and CAB,j are average surface concen-
tration of inactivated Product A on SAAA,i and
SAAB,j. The average surface concentrations
can be estimated from swab and rinse data as
described in Section AI.2 of Appendix I.
SBSA and SBSB are the smallest integral (7) batch
size of Product A and B, respectively
LDB and LDA are the largest doses of Products A
and B, respectively.
If inactivated Product A can be recovered by TOC
assay, then TOC is used to verify removal of the
inactivated product; otherwise, an alternative assay is
developed to demonstrate removal of the inactivated
product at full scale.
The CQ criterion can be applied to smaller sections
of the total equipment train. For instance, if the drug
substance (DS) and drug product (DP) are manufac-
tured in different facilities, it may be more efficient
from a change management perspective to decouple the
CQ assessments for the DS and DP equipment trains.
SETTING ACCEPTANCE LIMITS
FOR PROCESS RESIDUE
If the characterization data indicate that the API de-
grades when exposed to worst-case cleaning conditions,
54 Special edition: Cleaning Validation
and the CQ criterion per Equation 2 is met, then the
acceptance limit for the process residue can be set based
on the lowest of the following two limits:
Process capability. This is based on either the
capability of the equipment or the capability of
the CIP circuit to clean a specific process resi-
due. Note that the CQ approach is based on the
use of historical cleaning data to support the
baseline amount of inactivated Product A in a
dose of Product A. Thus, the use of historical
cleaning data to set acceptance limits for clean-
ing is justified. If the historical data are below
the limit of quantitation (LOQ) of the assay, the
process capability limit can be set to the LOQ of
the assay. This approach is consistent with indus-
try practices. It is justified because the cleaning
cycle is validated and continually monitored, and
has appropriate controls in place. Additionally,
adopting this approach does not impact the rela-
tive amount of degraded product that is carried
LD
over (i.e., M LD
IP AA vs. M IP AB), which is consis-
tent with the CQ criterion.
10 ppm limit for carryover of process residue.
This is a widely used default limit (8) that is
based on the criterion that the cumulative car-
ryover of process residue from the previously
manufactured batch into the subsequently manu-
factured batch should be 10 ppm. Note that
the raw data from the analytical instrument
should be divided by the appropriate swab or
rinse recovery factor when estimating the residue
on the surface. Additionally, if TOC is used to
analyze the sample, the default limit should be
multiplied by the fraction of carbon in the pro-
cess soil to account for the amount of carbon in
the residue.
CONCLUSION
An important consideration in multiproduct clean-
ing validation is to demonstrate that the carryover of
the previously manufactured API into a batch of the
subsequently manufactured product is below an accept-
able limit. If, however, the previously manufactured API
becomes pharmacologically inactive during cleaning,
then there is limited value in verifying removal of the
API. Instead, from a patient safety standpoint, it is more
meaningful to demonstrate that the inactivated product
is adequately removed.
Inactivation of the API during cleaning can be
assessed by exposing the process soil to simulated
cleaning conditions at bench scale. The degree of
inactivation is evaluated by subjecting the sample and
untreated controls to the appropriate assays (e.g., SDS-
PAGE and bioassay for evaluation of degradation and
biological activity, respectively).
 Rizwan Sharnez

If the protein is inactivated during cleaning, then Active Ingredients as Caused by Various Process
the acceptance limit for the inactivated degradants of Cleaning Agents and Temperature, Journal of Vali-
the previously manufactured product (Product A) in dation Technology, Vol 15, No. 3, p. 69, 2009.
the subsequently manufactured product (Product B) 4. Rathore, N., Qi, W., Chen, C., Ji, W., Bench-scale
can be set based on the CQ criterion. The CQ ap- characterization of cleaning process design space
proach provides assurance that the maximum amount for biopharmaceuticals, Biopharm Int, Vol 22, No.
of inactivated Product A that a patient taking Prod- 3, 2009.
uct B receives (M LD
IP AB) is limited to the maximum 5. Parental Drug Association, Inc., PDA Technical Report
amount of inactivated Product A that a patient taking 49 Points to Consider for Biotechnology Cleaning Vali-
Product A receives (M LD IP AA). The rationale for this dation, July 2010.
approach is that if one set of patients (i.e., those taking 6. Health Canada, Cleaning Validation Guidelines
Product A) are being exposed to a certain amount of (GUIDE-0028); Section 8.3, Health Products and
inactivated Product A, then it is acceptable for an- Food Branch Inspectorate, 2008.
other set of patients (i.e., those taking Product B) to 7. Note: i.e., before the original manufacturing batch is
be exposed to a lesser or equal amount of inactivated subdivided into smaller batches for processing (e.g.,
Product A. filling).
In terms of the mass of the inactivated product, the 8. Note: i.e., if the calculated acceptance limit is above
CQ criterion is given by Equation 1. the default limit, then the default limit is used; con-
In order to use this criterion to set acceptance limits versely, if the calculated acceptance limit is below
for inactivated product, the Equation 1 inequality the default limit, then the acceptance limit is used.
was expressed in terms of measureable equipment
and product parameters (Appendix I). The resulting ARTICLE ACRONYM LISTING
expression for the CQ criterion for intrasite assess- A Product A Previously manufactured prod-
ments, where the intracampaign cleaning cycle (AA) uct
is similar to the intercampaign cleaning cycle (AB), is AA Between batches of the same product (in-
given by Equation 2. tracampaign)
When this inequality is satisfied, the amount of AB Between batches of different products (inter-
inactivated Product A in the largest dose of Product B campaign)
will be less than the amount of inactivated Product A ADE Acceptable Daily Exposure
in the largest dose of Product A. API Active Pharmaceutical Ingredient
If inactivated Product A can be adequately recovered B Product B Subsequently manufactured
by TOC, then TOC is used to verify removal of the product
inactivated product; otherwise, an alternative assay is CQ Comparable Quality
developed to demonstrate removal of the inactivated DP Drug Product
product at full scale. DS Drug Substance
If the characterization data indicate that the API is LD Largest Dose
inactivated when exposed to cleaning conditions, and LOQ Limit of Quantitation
the CQ criterion is met, then the acceptance limits M LD
IP AA Mass of inactivated Product A in largest dose
for the process residue can be set based on the lowest of Product A
of the following two limits: process capability and 10 M LD
IP AB Mass of inactivated Product A in largest dose
ppm limit for carryover of process residue. of Product B
Application of the CQ criterion to intersite transfers MAC Maximum Allowable Carryover
will be described in Part III. MWD Molecular Weight Distribution
SDS PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel
REFERENCES Electrophoresis
1. Fourman, Gary L. and Michael V. Mullen, Deter- SBS Smallest Batch Size
mining Cleaning Validation Acceptance Limits for TOC Total Organic Carbon
Pharmaceutical Manufacturing Operations, Phar-
maceutical Technology, 17 (4), 54-60, 1993. ACKNOWLEDGEMENTS
2. Sharnez, R., To, A., Cleaning Validation of Multi- The authors thank Aine Hanly, Donna Corvese, Lillian
product Equipment: Acceptance Limits for Inac- Colon, Sam Guhan and Steve Hatke for their help and
tivated Product, Part IThe Comparable Quality support.
Approach, Journal of Validation Technology, Autumn
2011, p. 32-36, 2011.
3. Kendrick, K., Canhoto, A., Kreuze, M., Analysis
of Degradation Properties of Biopharmaceutical

Special edition: Cleaning Validation 55

Rizwan Sharnez

APPENDIX I facture Product A (Figure AI.2). The surface area that

Derivation of CQ Criterion
Derivation of the comparable quality (CQ) criterion in
terms of measureable equipment and product parameters
is described below.
In terms of the carryover of inactivated product, the
both products are exposed to, also known as the
shared surface area, is SA AB.
Similarly, the carryover of residual inactivated
Product A (that remains on the equipment surface after
batch
cleaning), into a batch of Product B (M IP,AB) can be
expressed as:
M

[C
batch
CQ criterion states that: M IP,AB = AB,j
SA AB,j] [Equation AI-2b]
The amount of inactivated Product A in the largest j=1
dose (LD) of Product B (M LD
IP AB) should be less than
or equal to the amount of inactivated Product A in the where, SA AB,j is the product contact surface area for
largest dose of Product A (M LD
IP AA) (2). Thus, the jth system of the equipment train (cm2), M is the
number of systems that are shared between the two
products, and CAB,j is the average surface concentration
M LD
IP AB M LD
IP AA [Equation AI-1] of inactivated Product A on SA AB,j (g/cm2).
Thus, the carryover of inactivated Product A into
AI.1 CQ criterion in terms largest dose of Product B (M LD
IP AB) can be expressed
of measureable parameters as:
N

In order to apply this criterion to set acceptance limits LDB
for inactivated product, the above inequality must be ex- M IP,AB=
LD
[CAB,j SA AB,j]
pressed in terms of measureable equipment and product j=1 SBSB
parameters. This is demonstrated in Figure AI.1 with an [Equation AI-3b]
example in which two products, A and B, are subse-
quently manufactured in a shared equipment train. where, LDB and SBSB are the largest dose and smallest
The equipment train shown in Figure AI.1 is used batch size of Product B, respectively.
to manufacture Product A and has a total surface area Substituting for the left-hand and right-hand sides
of SA AA. of Equation AI-1 with the right-hand side of Equations
The carryover of the residual inactivated Product A AI-3b and AI-3a, respectively, gives:
(that remains on the equipment surface after batchclean- M N

[C
LDB LDA
ing), into the next batch of Product A (M IP,AA) can be [CAB,j SA AB,j] AA,i
SA AA,i ]
SBSB SBSA
expressed as: j=1 i=1

[Equation AI-4]
N

[C
batch
M IP,AA = AA,i
SA AA,i] [Equation AI-2a] Rearranging the terms in Equation AI-4 gives:
i=1

N
[CAA,i SA AA,i ] SBSA LDB
where SA AA,i is the product contact surface area for the i=1


ith system (CIP circuit) of the equipment train (cm2), N M SBSB LDA
is the total number of systems that are used to manufac- j=1
[CAB,j SA AB,j ]
ture Product A, and CAA,i is the average surface concen- [Equation AI-5]
tration of inactivated Product A on SA AA,i (g/cm2).
Thus, the carryover of inactivated Product A into the Equation AI-5 gives the CQ criterion for intrasite
largest dose of Product A (M LD
IP AA) can be expressed as: manufacturing (i.e., when Product A is already being
manufactured at the same site where Product B is man-
N

LDA ufactured). Note that N is the total number of systems
M IP,AA=
LD
[CAA,i SA AA,i] used in the manufacture of Product A, and M is the
i=1 SBSA number of systems that are used in the manufacture of
both Product A and Product B (i.e., shared systems).
[Equation AI-3a]
AI.2. Surface concentration in terms of swab or rinse
where LDA and SBSA are the largest dose and smallest data
batch size of Product A, respectively. The surface concentration terms in Equation AI-5
Part or all of the equipment train that is used to (CAA,j and CAB,j) can be expressed in terms of TOC
manufacture Product B will also be used to manu- (or other analytical) results as follows:

56 Special edition: Cleaning Validation

 Rizwan Sharnez

Upstream Downstream Fill/Finish

Figure AI.1: Equipment train used to manufacture Product A has a total surface area of SA AA

Upstream Downstream Fill/Finish

Figure AI.2: The surface area that both products will be exposed to, also known as the shared surface area, is SAAB
S

1 swab recovery factor and carbon fraction for the process
CAA,i= CAA,k,i [Equation AI-6] soil in the ith system.
S k= 1 Similarly, CAA,k,i can be estimated from TOC rinse
data as follows:
where S is the number of data points (samples) for the
ith system (CIP circuit) and CAA,k,i are the measured
CAA,k,i =rTOCk,i
surface concentrations of inactivated product for each of [Equation AI-7b]
the S data points. rRFi cfi
The CAA,k,i for the equipment train used to manu-
facture Product A can be estimated from TOC swab where, rTOCk,i is the TOC rinse data for the kth rinse
data as follows: sample result for the ith system, and rRFi and cfi are the
rinse recovery factor and carbon fraction for the process
CAA,k,i =sTOCk,i soil in the ith system.
[Equation AI-7a] Similarly, CAB,j can be estimated from TOC swab
sRFi cfi or rinse data, recovery factors and carbon fractions for
the shared equipment.
where, sTOCk,i is the TOC swab data for the kth swab
sample result for the ith system, and sRFi and cfi are the

Special edition: Cleaning Validation 57

Rizwan Sharnez

APPENDIX II

N
i=1
[CAA,i SA AA,i ] SBSA LDB
Worked Example for Intrasite
SBSB LDA
M
Assessment
j=1
[CAB,j SA AB,j ]
Is the CQ criterion satisfied for the following scenario?:
Product A and Product B are currently being The calculated parameters for this example are as
manufactured at the same site. The total surface area given in Table AII-4.
associated with the manufacture of Product A and
3

[C
relevant process parameters are given in Table AII-1.
SA AA,i ]= (3.7112.6)+(2.1410.2)
TOC rinse data for the above circuits are listed in i=1
AA,i

Table AII-2. In this case, for each circuit there is one +(3.1511.6)=105.1
TOC rinse sample. 2

Part of the equipment train used to manufacture

Product A is also used to manufacture Product B. Cir-
[C
j=1
AB,j
SA AB,j] = (2.1410.2)+(3.1511.6)=58.4

cuits 2 and 3 are shared for the manufacture of Prod- SBSA 4.5
uct A and Product B. The shared surface area SA AB,j = = 0.9
SBSB 5
and relevant process parameters for the manufacturing
Product B are as given in Table AII-3. LDB 75
= = 1.5
Because the cleaning cycles for AA are the same as LDA 50
the cleaning cycles for AB, CAA,i = CAB,j for Cir-
cuits 2 and 3, and the TOC rinse data listed in Table Substituting the above results into the left and right
AII-2 can be used for both surface concentrations. hand sides of Equation AI-5 gives:
Left hand side = 1.80
AII. Solution Right hand side= 1.35
For this scenario (intrasite assessment), the CQ criterion Thus, the CQ criterion is satisfied for the above
is given in Equation AI-5: intrasite assessment. JVT

Table AII-3: Shared surface area for Products A and B and relevant process parameters.

Description of Parameter Abbreviation Value

Number of shared CIP circuits M 2
The shared surface area of circuit 2 (j=1) that is used to manufacture Prod-
SA A B,1 10.2 m 2
uct A and Product B
The shared surface area of circuit 3 (j=2) that is used to manufacture Prod-
SA A B,2 11.6 m 2
uct A and Product B
Smallest batch size of Product B SBSB 5 kg
Largest dose size of Product B LDB 75 mg

Table AII-4: Calculated parameters for intrasite assessment.

Calculated Parameter Equation Result Worked Example for i=1

C A A,1 AI-7b 3.71 ppm
C A A,2 AI-7b 2.14 ppm CAA,k,i =rTOCk,i
rRFi cfi
C A A,3 AI-7b 3.15 ppm
CAA,1,1=0.65 =3.71
C A B,1 C A B,1= C A A,2 2.14 ppm (0.70x0.25)

C A B,2 C A B,2=C A A,3 3.15 ppm

58 Special edition: Cleaning Validation

 Rizwan Sharnez

Table AII-1: Total surface area associated with the manufacture of Product A and relevant process parameters.

Description of Parameter Abbreviation Value

Number of CIP circuits N 3
Surface area of circuit 1 (i=1) that is used to manufacture Product A SA A A,1 12.6 m 2
Surface area of circuit 2 (i=2) that is used to manufacture Product A SA A A,2 10.2 m 2
Surface area of circuit 3 (i=3) that is used to manufacture Product A SA A A,3 11.6 m 2
Smallest batch size of Product A SBSA 4.5 kg
Largest dose size of Product A LDA 50 mg

Table AII-2: TOC rinse data.

Description of Parameter Abbreviation Circuit 1 (j=1) Circuit 2 (j=2) Circuit 3 (j=3)

TOC rinse sample data for the i circuit th
rTOCi,j 0.65 ppm 0.2 ppm 1.1 ppm
Rinse recovery factor for the i circuit
th
rRFj 0.70 0.55 0.92
Carbon fraction for the i circuit
th
cfj 0.25 0.17 0.38

Special edition: Cleaning Validation 59

Rizwan Sharnez

Biopharmaceutical
Cleaning Validation:
Acceptance Limits for
Inactivated Product
Based on Gelatin as a
Reference Impurity | IVT
Rizwan Sharnez, Ph.D.,Abby Spencer, Jeanine Bussiere, Ph.D., Dan Mytych, Ph.D., Angela
To, and Arun Tholudur, Ph.D.

Biopharmaceutical cleaning and sterilization processes denature and degrade

the active pharmaceutical ingredient (API)i into fragments that are pharmaco-
logically inactive. A rational approach for setting safety-based acceptance lim-
its for inactive fragments is described. The approach is based on the use of
gelatin as a reference impurity. It is designed to ensure that the carryover of
inactive fragments between batches of different products is acceptable from a
predictive safety standpoint.
The scope of this paper is limited to process residues of non-conjugated
human therapeutic proteins. Nonetheless, the underlying principles may be
useful in setting acceptance limits for other types of inactive impurities.

INTRODUCTION
An important regulatory expectation for multiproduct cleaning validation
is that the potential carryover of the previously manufactured API into the
subsequently manufactured product should be below an acceptable level.
This criterion is often met through a maximum allowable carryover (MAC)
assessment for the previously manufactured API (1-5). The MAC assessment
is typically based either on the minimum therapeutic dose (1), or the accept-
able daily exposure (ADE) (2) of the previously manufactured API.
A limitation of the conventional MAC approach is that it does not
provide appropriate acceptance limits for pharmacologically inactive
product. This has important implications for biopharmaceutical manu-
facturing because the API is inactivated during cleaning and sterilization.
Another limitation of this approach is that the acceptance limit for the
API is often below the process capability limit (PCL) of the cleaning pro-
cesses and, in some instances, below the limit of quantitation (LOQ) of
non-specific assays, such as total organic carbon (TOC). Other limitations
of the conventional MAC approach have been discussed previously (6).
Fragmentation and inactivation of an API during cleaning and steriliza-
tion can be assessed by exposing the process soil to simulated operating
conditions at bench scale (7). The bench scale experiments are designed
to simulate full-scale operating conditions that are least conducive
(worst-case) for inactivation. The degree of inactivation is evaluated by
subjecting the sample and untreated controls to the appropriate assays
(e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-
PAGE] and bioassay can be used to evaluate fragmentation and biological
activity, respectively). The results of the study are used to set appropriate

60 Special edition: Cleaning Validation


acceptance limits for the process residue. The product
inactivation approach is therefore more science-based
and reflective of the phenomenological aspects of
cleaning processes.
Inactivation of the product during cleaning and
sterilization has important implications for clean-
ing validation of multiproduct equipment. If the API
degrades into pharmacologically inactive fragments,
the acceptance limit for the process residue can be set
based on the inactive fragments instead of the active
ingredient. This is consistent with the expectation that
the carryover of an impurityin this case the inac-
tive fragmentsinto the subsequently manufactured
product should be justified from the standpoint of the
safety and efficacy of the product.
The experimental approach and analytical methods
for assessing fragmentation and inactivation of the API
have been discussed in the literature (6-11). This paper
describes a rational approach for setting safety-based
acceptance limits for the inactive fragments. The pro-
posed methodology builds upon previously published
approaches (6, 10-11).
ASSESSING THE SAFETY OF
INACTIVE FRAGMENTS OF HTPS
The safety of inactive fragments of human therapeutic
proteins (HTPs) is assessed qualitatively in this section.
Consider an equipment train that is used to manufac-
ture Product A. The cleaning and sterilization cycles are
known to denature and degrade any residual product in
the equipment into fragments that are pharmacologically
inactive. The inactive fragments of Product A are carried
over into the subsequent batch of Product A. Thus, as
a class of molecules, inactive fragments of HTPs do not
present a new or unknown risk from a safety standpoint.
In fact, these types of fragments have been present in
biopharmaceutical products for decades. Further, a com-
prehensive literature search did not reveal any evidence
of safety or efficacy issues directly attributable to the
presence of inactive fragments in parenteral drugs (12).
Implications for Multiproduct Cleaning Validation
Consider the introduction of a new product (Product B)
into the facility. Part of the equipment train is now used
to process both products. The cleaning and sterilization
cycles between batches of different products (A B or B
A, [i.e., intercampaign processing or changeover]) are
the same as those between batches of the same product
(A A or B B, [i.e.,intracampaign processing]). Thus,
for a given set of cleaning and sterilization cycles, the
molecular weight distribution (MWD) of inactive frag-
ments of Product A (IFA) that are carried over into a sub-
sequent batch of Product B (intercampaign processing) is
the same as the MWD of IFA that are carried over into a
subsequent batch of Product A (intracampaign process-
ing). The same is true for the MWD of inactive frag-
Rizwan Sharnez
ments of Product B (IFB). Thus, the IFA that are carried
over during changeover into Product B do not present
a new or unknown risk from a safety standpoint. This
implies that the equipment train can be used to manu-
facture multiple products without introducing a new or
unknown class of impurities into any of the products.
Further, the carryover of IFA into Product B is significant
only for the first lot of Product B that is manufactured
following changeover.
COMPARABLE QUALITY APPROACH
The acceptance limits for inactive fragments in the pro-
cess residue can be set based on the Comparable Quality
(CQ) approach (6, 10-11). With the CQ approach, the
amount of the target impurityin this case inactive frag-
ments of Product Athat is carried over into the largest
dose (i.e., largest dose that is administered to a patient
in a day) of the subsequently manufactured product
(Product B) is limited to the acceptable exposure per
dose of a reference impurity. The reference impurity must
be comparable to or worse than the target impurity from
a predictive safety standpoint.
Predictive safety for inactive fragments is evaluated
in terms of the key factors that determine toxicity and
immunogenicity. For HTPs, toxicity is determined by
pharmacological activity (13); thus, toxicity is generally
not a concern for inactive fragments of HTPs. Im-
munogenicity is primarily determined by foreignness
and chemical complexity (14). Chemical complexity
increases with molecular weight (MW); thus, larger
molecules tend to be more immunogenic (15). The
most active immunogens tend to have a MW greater
than 100 kilo Daltons (kDa) (14). HTP fragments with
MWs less than 10 kDa are generally weak immuno-
gens (14). Small polypeptides under 10 kDa usually
need to be conjugated to large immunogenic carrier
proteins or administered with adjuvants to ensure an
antibody response (16).
The suitability of gelatin as a reference impurity for
setting acceptance limits for inactive HTP fragments is
evaluated in the next section.
GELATIN AS A REFERENCE IMPURITY FOR
INACTIVE HTP FRAGMENTS
The use of gelatin as a reference impurity for inactive
HTP fragments is justified for the following reasons:
Gelatin consists of a mixture of animal protein frag-
ments derived from the hydrolysis of collagen, a pro-
tein that is commonly found in connective tissues
(17). The collagen is hydrolyzed by exposing the
connective tissues to pH and temperature extremes
(18). HTPs in the process residue are exposed to
similar operating conditions during cleaning and
sterilization. Thus, in terms of chemical composi-
tion, the protein fragments in gelatin are comparable
to the HTP fragments in the process residue.
Special edition: Cleaning Validation 61
Rizwan Sharnez

To elicit an immune response, a molecule must
be recognized as nonself by the immune system
(14). The protein fragments in gelatin are of ani-
mal origin whereas the HTP fragments in the
process residue are of human origin. Thus, the
peptide sequences in the HTP fragments are more
likely to be recognized by human immune sys-
tems than the peptide sequences in the protein
fragments in gelatin. Consequently, as compared
to the protein fragments in gelatin, the HTP frag-
ments in the process residue are less likely to
elicit an immunogenic response in humans.
The molecular weights of most of the HTP frag-
ments are less than 10 kDa (19). HTP fragments
with MWs less than 10 kDa are generally weak
immunogens (14). Protein fragments in gelatin
range from 15 kDa to 400 kDa (18), which is
substantially higher than the 10 kDa threshold
for weak immunogens.
CQ APPROACH BASED ON GELATIN
This section describes the application of the CQ ap-
proach based on the use of gelatin as a reference impu-
rity to inactive HTP fragments.
Gelatin is used as a stabilizer in many parenteral
products. The amount of gelatin in common parenteral
products ranges from several hundred micrograms to
over 15,000 g per dose (refer to table).

Table: Gelatin Content of Common Parenteral Products.

Product Trade Name Gelatin Content Route of Dosing Schedule

per dose Administration
DTaP (Sanofi Pasteur)1, 2 Tripedia 28 g in 0.5 mL Intramuscular Five dose series at two, four,
injection six, 15 months, and four to six
years of age
Influenza (Sanofi Pasteur)2 Fluzone 250 g in 0.5 mL Intramuscular Yearly
injection
Japanese Encephalitis JE-VAX 500 g in 1.0 mL Subcutaneous Three doses on days zero,
(Sanofi Pasteur)2 injection seven, and 30. Booster dose of
1 mL can be given after two
years
Leuprolide acetate for Lupron Depot, 650 g in 3.75 mg Intramuscular Monthly
depot suspension (Abbot)3 3.75 mg

Measles, Mumps, Rubella ATTENUVAX 14,500 g in 0.5 mL Subcutaneous Two doses: one at 12 months of
(Merck)2 injection age and one at four years of age
Measles, Mumps, Rubella, ProQuad 11,000 g in 0.5 mL Subcutaneous Two doses: one at 12 months of
Varicella (Merck)2 injection age and one at four years of age
Rabies (Novartis)2 RabAvert 12,000 g in 1.0 mL Intramuscular Post-exposure dosage: 1 mL
injection doses on days zero, three,
seven, 14, and 28
Varicella (Oka/Merck)2 VARIVAX 12,500 g in 0.5 mL Subcutaneous Two doses each given four
(frozen) injection weeks apart
Yellow Fever (Sanofi YF-VAX 7,500 g in 0.5 mL Subcutaneous One dose every 10 years
Pasteur)1, 2 injection
Zoster (Oka/Merck)2 ZOSTAVAX 15,580 g in Subcutaneous Single dose
0.65 mL injection
Isoplex Solution for ISOPLEX 4% w/v (20g in Intravenous
Given for blood loss, 500 mL
Infusion4 500 mL bag) can be given in as little time as
five minutes in the case of rapid
blood loss
1
Kelso, John M., Li, James T., Adverse reactions to vaccines; Ann Allergy Asthma Immunol., 2009 Oct;103(4 Suppl
2):S1-14.
2
Package inserts via www.fda.gov/BiologicsBloodVaccines/Vaccines/default.htm
3
Package insert via http://www.rxabbvie.com
4
Package insert via http://www.mhra.gov.uk/Safetyinformation/Medicinesinformation/SPCandPILs/index.htm

62 Special edition: Cleaning Validation

 Rizwan Sharnez

Lupron serves as a good model for the CQ approach SC SSA /

IFA
[6b]
because it is administered in multiple doses over an
extended period. Note that from the standpoint of Rearranging Equation 6b gives
immunogenicity, repeated dosing of an immunogen
is important; however, dosing frequency is generally SC 0.65 mg (
IFA
/ )/ SSA [6c]
not critical provided that the time interval between
exposures is relatively short (e.g., weekly, bi-weekly, The above inequality can be used to set an ac-
or monthly). Each dose of Lupron contains 0.65 mg of ceptance limit forSC in terms of three measureable
IFA

gelatin; thus, the CQ Criterion based on the gelatin in parameters:

this product is , , and SSA.

0.65 mg[1a] Solved Example

Calculate the acceptance limit for TOC in the swab ex-
Where re
is the mass of inactive frag- tractant for the following scenario: = 20 L, =
ments of Product A (IFA) that is carried over into the 10 mL, SSA = 105 cm2, swab recovery factor for the pro-
largest dose (LD) of the subsequently manufactured cess residue (SRF) = 0.5, carbon fraction in the process
Product (B). residue (CF) = 0.5, area swabbed (A) = 25 cm2, and mass
The concentration of the inactive fragments of Prod- of extractant (m) = 25 g.
uct A (CIFA) in the largest dose of Product B ( ) is, Substituting for , ,and SSA into Equation
6c gives:
CIFA=
[2a]

SC 650 (g) 20,000 (mL) / 10 mL / 105 cm2 =
IFA

13 g/cm2
Substituting for from Equation 1a gives Based on the worst-case assumption that all the
the acceptance limit (AL) for CIFA, carbon in the process residue is from the IFA, the ac-
ceptance limit for TOC (ALTOC) in the swab extractant
[2b]
is
ALTOC = SC A SRF CF / m
IFA

A worst-case estimate of CIFA ( ) is obtained by ALTOC = 13 g/cm2 25 cm2 0.5 0.5 / 25 g

assuming that all of the residual IFA on the surface = 3.25 ppm
of the shared equipment train after cleaning (RIFA) is Thus, the cleaning is effective if the TOC in the
transferred into the smallest batch of Product B ( swab extractant is 3.25 ppm above the baseline TOC
) that is manufactured in the equipment trainii. Thus, of the control sample.
Note that the above acceptance limit is based on
=R / [3] relatively unfavorable system parameters (small batch
size, large dose and shared surface area, and low
Also, recovery). Further, the acceptance limit is considerably
higher than the process capability limit (PCL) of most
RIFA = SSA SCIFA [4] cleaning processes, which is typically on the order of 1
ppm Carbon for TOC swab samples. It is also substan-
Where SSA is the shared surface area of the equip- tially higher than the LOQ of TOC, which is typically
ment train, andSC is the average surface concentra-
IFA
between 0.05 and 0.2 ppm Carbon. It is therefore un-
tion of IFA on the shared surface area. Thus, likely that the acceptance limit based on this approach
would be below the PCL of the cleaning process or the
=SC SSA/
IFA
[5] LOQ of TOC.

Note that any splits of the batch into smaller batches METHODOLOGY FOR SETTING ACCEPTANCE LIMITS
for filling or other operations should be appropriately The proposed methodology for setting acceptance
accounted for in estimating SSA and . limits for multiproduct equipment is summarized in
Based on the definitions of andC ALIFA, the the flowchart. Inactivation studies are first performed
cleaning is effective if under simulated worst-case operating conditions (7). If
the sample is shown to have no detectable activity, the
[6a] CQ approach can be used to set acceptance limits for the
inactivated product (right side of flowchart). However,
Substituting for and from Equation 2b if there is no detectable loss in activity, the conventional
and Equation 5, respectively, gives MAC approach can be used to set the acceptance limit

Special edition: Cleaning Validation 63

Rizwan Sharnez
Figure 1: Flowchart for Setting Acceptance Limits (AL) for the Process Residue.
a
Based on results of bioassay
for the previously manufactured product (1-2). If the
results indicate that the API is partially inactivated, the
acceptance limit should be determined for the API as
well as the inactivated product, and the lower of the
two limits should be used. Alternatively, the operating
parameters can be modified to ensure inactivation of the
API. This can be facilitated by running additional studies
to characterize the effect of the operating parameters on
the API.
Default Limits
If the carryover of the target impurity into the subse-
quent batch based on the above acceptance limit (AL) is
above 10 ppm, then the AL should be reduced to limit
the carryover to 10 ppm. The 10 ppm carryover limit is a
widely used default limit (20). It is based on the empiri-
cal observation that raw materials and intermediates
commonly found in biopharmaceutical manufacturing
process residues are safe to ingest at concentrations up to
0.1% (1000 ppm). In order to extrapolate this oral limit
to other routes of administration, it is reduced by a factor
of 100 to 10 ppm.
If sufficient historical cleaning data are available to
establish a statistically sound process capability limit
(PCL), then the AL should be set to the PCL. If the
historical data are below the LOQ of the analytical
method, the PCL can be set to the LOQ of the analyti-
64 Special edition: Cleaning Validation
cal method. This approach is consistent with industry
practices and is justified because the cleaning cycle is
validated and continually monitored, and has appro-
priate controls in place. Additionally, adopting this ap-
proach does not impact the relative amount and MWD
of the target impurity that is carried over between
batches of different products, which is consistent with
the CQ criterion. Thus, setting the acceptance limit to
the PCL ensures that the MWD of the target impurity
that is carried over into a batch of Product B is com-
parable to the MWD of that target impurity that was
historically present in a batch of Product A.
CONCLUSION
Biopharmaceutical cleaning and sterilization cycles are
designed to expose product contact equipment to pH
and temperature extremes for several minutes. Under
these conditions biological APIs degrade and denature
rapidly thereby becoming pharmacologically inactive.
Inactivation of the product during cleaning and
sterilization has important implications for cleaning
validation of multiproduct equipment. If the API is in-
activated, the acceptance limits for the process residue
can be set based on the inactive product instead of the
API.
A comprehensive literature search did not reveal any
evidence of safety or efficacy issues directly attribut-

able to the presence of inactive fragments in parenteral
drugs (12). Further, these types of fragments have been
present in biopharmaceutical products for decades.
Thus, as a class of molecules, inactive fragments of
HTPs do not present a new or unknown risk from a
safety standpoint.
The Comparable Quality (CQ) approach based on
gelatin can be used to set acceptance limits for inactive
fragments of non-conjugated HTPs. With this ap-
proach, the carryover of inactive fragments into the
largest dose of the subsequently manufactured product
is limited to the acceptable exposure of an appropri-
ate reference impurity. The reference impurityin this
case gelatinwas shown to be comparable to or worse
than the inactive fragments from a predictive safety
standpoint.
If the product is not inactivated during cleaning
and sterilization, the acceptance limit for the process
residue should be set based on the acceptable carry-
over of the API (1-2). However, if the results indicate
that the API is partially inactivated, the acceptance
limits should be determined for the API as well as for
the inactive fragments, and the lower of the two limits
should be used for cleaning validation (refer to Figure).
The acceptance limit for inactive fragments based
on gelatin as a reference impurity was ascertained to
be 0.65 mg per dose. At 0.65 mg of inactive fragments
per dose, the acceptance limit for TOC swab samples
was shown to be 3.25 ppm Carbon.iii This estimate
was based on relatively unfavorable system parameters.
Note that this acceptance limit is substantially higher
than the LOQ of TOC, which is typically between
Rizwan Sharnez
0.05 and 0.2 ppm Carbon. It also compares favorably
to the process capability limit (PCL) of most cleaning
processes, which is typically on the order of 1 ppm
Carbon for TOC swab samples. Thus, with the CQ ap-
proach based on gelatin, it is unlikely that the accep-
tance limit for the process residue would be below the
PCL of the cleaning process or the LOQ of TOC.
The methodology described in this paper is not
applicable to allergenic ingredients, penicillin, cepha-
losporin, potent steroids, and cytotoxic compounds.
Acceptance limits for process residues associated with
these types of APIs are typically set to the limit of
detection (LOD) of the best available analytical method
(21).
i
Depending on the process soil, API refers to the
active pharmaceutical ingredient in the drug product,
drug substance, or drug substance intermediate.
ii
i.e., surface area of equipment train that is subjected
to cleaning and that comes into contact with both
Product A and Product B. If inactive fragments of
Product A are removed by the purification steps
for Product B, then the part of the equipment train
upstream of those purification steps can be excluded
from the shared surface area.
iii
Note that the use of a non-specific method such as
TOC also allows for the detection of intact protein.
ACKNOWLEDGEMENTS
We thank Vijay Chiruvolu, Sam Guhan, Aine Hanly and
Anthony Mire-Sluis for their help and support.
Special edition: Cleaning Validation 65
Rizwan Sharnez

ACRONYMS
A Area swabbed
AA Between batches of the same product (intracampaign)
AB Between batches of different products (intercampaign)
ADE Acceptable Daily Exposure
AL Acceptance limit
ALCQ Acceptance limit based on comparable quality approach
ALMAC Acceptance limit based on MAC
ALTOC Acceptance limit for TOC swab sample
API Active pharmaceutical ingredient
Acceptable limit for concentration of inactivated fragments of Product A
CIFA Concentration of inactivated fragments of Product A
Worst-case estimate of CIFA
CF Carbon fraction in process residue
CQ Comparable quality
HTP Human therapeutic proteins
IFA Inactive fragments of Product A
IFB Inactive fragments of Product B
kDa Kilo Dalton
LD Largest dose
LOD Limit of detection
LOQ Limit of quantitation
m Mass of extractant
MAC Maximum allowable carryover
Mass of inactive fragments of Product A in largest dose of Product B
MW Molecular weight
MWD Molecular weight distribution
PCL Process capability limit
PR Process residue
Product A Previously manufactured product
Product B Subsequently manufactured product
RIFA Residual IFA on the surface of the shared equipment train after cleaning
SC IFA
Average surface concentration of IFA on the shared surface area
SDS PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SRF Swab recovery factor
Shared surface area (i.e., surface area of the equipment train that comes into contact with
SSA
both Product A and Product B)
TOC Total organic carbon
Largest dose of Product B

Minimum volume of the final batch of Product B (i.e., smallest batch size of Product B)

66 Special edition: Cleaning Validation

 Rizwan Sharnez and Angela To

Multiproduct Cleaning
Validation: Acceptance
Limits for the Carryover
of Inactivated API
Part IThe Comparable
Quality Approach
Rizwan Sharnez and Angela To

New Perspectives on Cleaning is an ongoing series of articles dedicated

to cleaning process development, validation, and monitoring. This column
addresses scientific principles, strategies, and approaches associated with
cleaning that are faced in everyday work situations.
Reader questions, comments, and suggestions are requested for future
discussion topics. These can be submitted to the column coordinator Rizwan
Sharnez at rsharnez@amgen.com.

SUMMARY
An important consideration in multiproduct cleaning validation is to dem-
onstrate that the carryover of the previously manufactured active pharma-
ceutical ingredient (API) into a batch of the subsequently manufactured
product is below an acceptable limit. If, however, the previously manufac-
tured API becomes therapeutically inactive during cleaning, then there is
limited value in verifying clearance of the API. Instead, it is more appropri-
ate to demonstrate clearance of inactivated product. This approach is gaining
acceptance in the industry.
A methodology for evaluating the degree of inactivation of a product
during cleaning and setting acceptance limits for the carryover of inacti-
vated product in multiproduct equipment is described. A new approach
for justifying acceptance limits for inactivated product, known as the
comparable quality (CQ) approach, is described in Part I; the application
of this approach to biopharmaceutical cleaning will be described in Part
II. The general principles of the CQ approach are applicable to most ac-
tive pharmaceutical ingredients (APIs).

INTRODUCTION
For multiproduct cleaning validation, acceptable carryover of the previously
manufactured API (Product A) into the subsequently manufactured API
(Product B) is determined through a maximum allowable carryover (MAC)
calculation (1-3). A limitation of the conventional MAC approach is that
it does not account for the carryover of the inactivated molecule between
lots of different products (i.e., A B, or B A). This is an important factor
to consider when aggressive cleaning conditions are used. For example,
biopharmaceutical cleaning cycles are generally designed to expose prod-
uct contact equipment to extremes of pH (i.e., 2-13) and temperature (i.e.,
60-80C) for several minutes. The equipment may also be steam sterilized
or sanitized after cleaning. Under these conditions, monoclonal antibod-

Special edition: Cleaning Validation 67

Rizwan Sharnez and Angela To
ies, therapeutic proteins, and other biological APIs are
known to degrade and denature rapidly (4, 5) and are,
therefore, likely to become therapeutically inactive (6).
Inactivation of the product during cleaning has
important implications for cleaning validation of mul-
tiproduct equipment. If it can be demonstrated that
the product becomes therapeutically inactive during
cleaning, a MAC assessment for the API would not be
required. It also obviates the need to develop product
specific assays (PSA) for cleaning validation.
Inactivation of the product during cleaning can be
assessed by exposing the process soil to simulated
cleaning conditions at bench scale (4, 5). The bench
scale studies are designed to simulate the conditions
that are least conducive (worst-case) for inactivation. For
example, for alkaline washes, the lowest applicable pH,
temperature, duration, and ratio of cleaning solution
to process soil is used to simulate the cleaning cycle at
bench scale. The sample is then neutralized and cooled
to minimize any further inactivation. The degree of in-
activation is evaluated by subjecting the sample and an
untreated control to the appropriate assays (e.g., Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
[SDS PAGE] and bioassay for biological products).
A literature search did not uncover any scientific
approaches or regulatory guidances for setting accept-
able limits for the carryover of inactivated product for
cleaning validation.
LIMITATIONS OF THE MAC APPROACH
The MAC approach is often used to set cleaning valida-
tion acceptance criteria for the carryover of the previous-
ly manufactured API (Product A) into the subsequently
manufactured API (Product B) (1-3). A limitation of this
approach is that it does not account for the carryover of
the inactivated molecule between lots of different prod-
ucts (i.e., A B, or B A).
Another limitation of the MAC approach is that the
acceptance limits for cleaning validation are often below
process capability limits and/or below the limit of quan-
titation (LOQ) of non-specific assays (e.g., total organic
carbon [TOC]; the LOQ of TOC is typically between
0.05 and 0.2 ppm). This issue is further exacerbated
by the low recovery of APIs from process soils. PSAs
such as enzyme-linked immunosorbent assay (ELISA)
and enzyme immunoassay (EIA) are sometimes used
to address this issue because they have very low LOQs
(typically below 10 ppb). However, PSAs are difficult
and laborious to qualify, and can give inaccurate results
if the API degrades during cleaning (7). That is because
PSAs detect activity indirectly, by recognizing specific
epitopes (i.e., short amino acid sequences that PSAs are
designed to detect). The epitopes can be destroyed by
buffer and cleaning agent components. However, a bio-
logical API can be therapeutically active even if the epi-
topes are destroyed, and this can lead to false negatives.
68 Special edition: Cleaning Validation
Similarly, it is possible for the API to be inactivated even
though the epitopes are not completely destroyed, and
this can lead to false positives. Another issue with PSAs
is that it is difficult to get an accurate recovery factor.
That is because the experimental conditions of the
recovery study (e.g., direct spotting of coupons with the
API) do not represent the actual sample matrix (i.e., a
small amount of API in the presence of degradants and
cleaning agent residues). For the above reasons, PSAs
should be used judiciously for verifying clearance of
biological APIs after cleaning (7).
Another issue with the MAC approach is that every
time a new product is introduced into a facility there is
a risk that one or more of the new MAC limits for the
previously validated products could be below the exist-
ing acceptance limits for cleaning validation.
PROTEIN DEgRADATION
AND INACTIvATION APPROACH
Performing Degradation and Inactivation Studies
A bench-scale approach for evaluating the bioactivity of
the residual API and the molecular weight distribution of
the degradants after exposure to cleaning conditions is
described in this section.
Full-scale cleaning conditions of shared product
contact equipment are evaluated to determine the con-
ditions that are least conducive for inactivation (e.g.,
lowest ratio of wash volume to protein, lowest cleaning
agent concentration, lowest temperature, and shortest
duration of exposure). The product is exposed to these
conditions at bench scale. The objective of the study is
to ascertain whether the API in the process sample is
inactivated when exposed to cleaning conditions.
Product is spiked into tubes containing alkaline
cleaning solution, heated to the appropriate tempera-
ture, and allowed to incubate for the duration of the
alkaline wash. Samples may also be titrated with the
acidic cleaning solution to the pH of the acidic wash
and held for the duration of the acidic wash. Samples
are then titrated to a neutral pH and cooled to 4C to
minimize further degradation and inactivation. The
samples are then subjected to SDS-PAGE and bioas-
say to determine the degree of degradation and TOC
analysis to determine whether the degradants can be
adequately recovered by TOC.
Assays
SDS-PAGE and bioassays are used to evaluate protein
degradation and inactivation, respectively. The product
is exposed to cleaning conditions at small scale and then
analyzed with the above assays to determine degree of
degradation (i.e., molecular weight distribution) and
bioactivity. Additionally, samples are analyzed for TOC
to determine the recovery of the inactivated protein and
the applicability of the TOC assay for demonstrating
clearance of the inactivated product at full scale.

SDS-PAGE solubilizes aggregated and degraded
proteins and separates them based on molecular
weight (MW). The inclusion of MW standards allows
for estimation of the MWs of the degradants and any
aggregates, and the inclusion of control samples at a
defined protein load allows for the estimated quantita-
tion of protein concentration by densitometry. Staining
of gels is sensitive to 5-10 ng for Silver Staining and
100 ng for Coomassie Staining. SDS-PAGE has the
advantage of providing a wide range of specificity for
detecting proteins with unknown primary structures,
size, charge, and hydrophobic states. This feature is
particularly useful for protein degradation analysis
because the level of protein degradation due to clean-
ing is highly unpredictable and can extend over a wide
range of MW. SDS-PAGE also provides high sensitivity
for detecting trace amounts of protein.
Bioassays measure the relative amount of biological-
ly active product present in a sample. Thus, bioassays
can be used to determine the effect of cleaning condi-
tions on the inactivation of biologicals.
SETTINg ACCEPTANCE LIMITS
BASED ON PROTEIN INACTIvATION
The methodology for setting acceptance limits is summa-
rized in the Figure. Degradation and inactivation studies
are first performed under simulated cleaning conditions.
If the sample is shown to have therapeutically active
Figure: Methodology for setting acceptance limits flowchart. Continued on page 70
Inputs
Process samples and
reference standard
Cleaning cycle and
equipment parameters
sample inactivated after
1 Based on
results of
bioassay
Yes
Use CQ approach to
set acceptance limit
(AL) for inactivated
API
continued
Rizwan Sharnez and Angela To
product, the MAC approach is used to limit carryover of
previous product to an acceptable level (1-3). If the MAC
limit is higher than the LOQ of TOC, TOC is used to
verify clearance of previous product at full scale. If the
MAC limit is below the LOQ of TOC, an alternate assay
may need to be developed to verify clearance of the API.
The MAC approach limits the amount of API of the
previously manufactured product (A) in a dose of the
subsequently manufactured product (B) to the accept-
able daily exposure (ADE) of A (8), or to 1/1000th of
the minimum dose of A (9).
COMPARABLE QUALITY APPROACH
If the protein becomes therapeutically inactive during
cleaning, then the acceptance limit for the inactivated
molecule of the previously manufactured product
(Product A) in the subsequently manufactured product
(Product B) can be set based on the CQ approach. With
the CQ approach, the amount of inactivated Product A in
a dose of Product B is limited to the amount of inacti-
vated API of Product A in a dose of Product A. Appropri-
ate adjustments are made to account for differences in
process parameters of the two products. If inactivated
Product A can be recovered and detected by TOC, and
its acceptance limit is greater than the LOQ of TOC, then
TOC is used to verify clearance of the inactivated API.
Otherwise, an alternative assay is developed to demon-
strate clearance of the inactivated API at full scale.
Perform
inactivation
study
Is API in process
exposure to cleaning
conditions?1
No
Use MAC
approach to
set acceptance
limit (AL) for API
continued
Special edition: Cleaning Validation 69
Rizwan Sharnez and Angela To

Continued from page 69

Inputs
Dose and batch sizes of
Use MAC
products
Surface area and rinse
approach to
parameters of shared set acceptance
equipment limit (AL) for API
Recovery study for API
Carbon content of API

Is AL LOQ
of TOC?
Yes No

Develop alternative
Use TOC to
assay to
demonstrate demonstrate
clearance of API
clearance of API

Inputs
Dose and batch sizes of products Use CQ approach to set
Surface area of dedicated and
acceptance limit (AL) for
shared equipment
Recovery study for inactivated API inactivated API
Carbon content of inactivated API

Can
inactivated API
be recovered
by TOC?
Yes No

2 Thisis
No done on a
Is AL LOQ
case-by-
of TOC?
case basis
Yes
Use TOC to
Develop alternative assay
demonstrate
to demonstrate clearance
clearance of
of inactivated API2
inactivated API

70 Special Edition: Cleaning Validation

 Rizwan Sharnez and Angela To

CONCLUSION ACKNOWLEDgEMENTS
The inactivation of a product during cleaning and steam- The authors are grateful to Arun Tholudur and Joel
ing has important implications for cleaning validation Bercu for their helpful suggestions and support.
of multiproduct equipment. Demonstrating that the
product becomes inactivated during these operations ob- ARTICLE ACRONYM LISTINg
viates the need to perform arduos MAC assessments for A Product Apreviously manufactured product
the API. It also eliminates the need to develop PSAs for ADE Acceptable Daily Exposure
cleaning validation. PSAs are designed to detect specific AL Acceptance Limit
epitopes; thus, if the API degrades, the result from the as- API Active Pharmaceutical Ingredient
say may not necessarily correlate to therapeutic activity. B Product Bsubsequently manufactured
The CQ approach can be used to set acceptance lim- product
its for the carryover of inactivated product for multi- CQ Comparable Quality
product cleaning validation. This approach is designed EIA Enzyme Immunoassay
to ensure that the amount of inactivated Product A in ELISA Enzyme-Linked Immunosorbent Assay
a dose of Product B is less than the amount of inacti- LOQ Limit of Quantitation
vated Product A in a dose of Product A. Application of MAC Maximum Allowable Carryover
the CQ approach to biopharmaceutical cleaning will MW Molecular Weight
be described in Part II of this series. PSA Product Specific Assay
SDS PAgE Sodium Dodecyl Sulfate Polyacrylamide Gel
REFERENCES Electrophoresis
1. Sharnez, R., Strategies for Setting Rational MAC- TOC Total Organic Carbon
based LimitsPart I: Reassessing the Carryover
Criterion, Journal of Validation Technology, Vol 16,
No. 1, p. 71-74, 2010.
2. Sharnez, R., To, A., Klewer, L., Strategies for
Setting Rational MAC-based LimitsPart II: Ap-
plication to Rinse Samples, Journal of Validation
Technology, Vol 17, No. 2, p. 43-46, 2011.
3. Sharnez, R., To, A., Strategies for Setting Rational
MAC-based LimitsPart III: Leveraging Toxicol-
ogy and Cleanability Data, Journal of Validation
Technology, Vol 17, No. 3, p. 24-28, 2011.
4. Kendrick, K., Canhoto, A., Kreuze, M., Analysis
of Degradation Properties of Biopharmaceutical
Active Ingredients as Caused by Various Process
Cleaning Agents and Temperature, Journal of Vali-
dation Technology, Vol 15, No. 3, p. 69, 2009.
5. Rathore, N., Qi, W., Chen, C., Ji, W., Bench-scale
characterization of cleaning process design space
for biopharmaceuticals, Biopharm Int, Vol 22,
No. 3, 2009.
6. Martinez, J.E., Immunogenic Potential of Thera-
peutic Protein Residues after Cleaning, Bioprocess
International, Vol. 9, P. 38-44, 2004.
7. Health Canada, Cleaning Validation Guidelines
(GUIDE-0028); Section 8.3, Health Products and
Food Branch Inspectorate, 2008. http://www.hc-sc.
gc.ca/dhp-mps/compli-conform/gmp-bpf/valida-
tion/index-eng.php
8. ISPE, Risk-Based Manufacture of Pharmaceutical
Products: A Guide to Managing Risks Associated with
Cross-Contamination, 1st Edition, Vol. 7, ISPE, 2010.
9. Fourman, Gary L. and Michael V. Mullen, Deter-
mining Cleaning Validation Acceptance Limits for
Pharmaceutical Manufacturing Operations, Phar-
maceutical Technology, 17 (4), 54-60, 1993. JVT

Special edition: Cleaning Validation 71

Sebastian Clerkin

Validation of a Cleaning
Process for Medical
Devices | IVT
Sebastian Clerkin, Ph.D.

AbStrACt
Many medical device manufacturers find it a considerable challenge
to plan and conduct a cleaning validation. The main challenges are
an establishment of the cleanliness limits and an identification of the
challenge conditions to be assessed during the process validation. This
paper describes a logical risk-based approach to overcome these chal-
lenges.
It begins with assessing the complete manufacturing process and
identifying the manufacturing agents. It discusses risk tools to deter-
mine which manufacturing agents need to have cleanliness limits. It
describes the manufacturing conditions to be considered when con-
ducting the cleaning validation.
The concepts described within this paper can be utilized by a medi-
cal device manufacturer to establish a cleaning process that will con-
sistently provide clean medical devices and comply with the relevant
regulations.

INtrODUCtION
Contamination of a medical device can have serious implications. Medi-
cal device manufacturers must ensure they have correctly identified all
potential contaminants and have established controls.
The United States Food and Drug Administration captures this re-
quirement within the Quality System Regulations (QSR) by stating that
each manufacturer shall (1, 2):
Establish and maintain procedures to prevent contamination of product by
substances that could be expected to have an adverse effect on product quality
Establish and maintain procedures for the use and removal of manufactur-
ing materials to ensure that it is removed or limited to an amount that does
not adversely affect the devices quality (2).
International Organization for Standardization (ISO) 13485 requires
that a medical device manufacturer establish documented requirements
for the cleanliness of a medical device in the following circumstances
(3):

Product is cleaned by the organization prior to sterilization and/or

its use, or
Product is supplied non-sterile to be subjected to a cleaning process
prior to sterilization and/or its use, or
Product is supplied to be used non-sterile and its cleanliness is of
significance in use, or
Process agents are to be removed from product during manufac-
ture.

Therefore, to comply with the QSR and ISO 13485, a medical device
manufacturer must establish documented cleanliness requirements.

72 Special edition: Cleaning Validation


Figure 1: Example of a Manufacturing Process Flow.
However, these regulations do not explicitly state
that a cleaning process validation must be completed.
The Global Harmonization Task Force (GHTF) Study
Group 3 does provide guidance on the requirement
for a cleaning process validation. Their process vali-
dation guidance (4); which was written by regula-
tors in the US, Europe, Japan, Australia and Canada;
states a cleaning process may be validated or may
be satisfactorily covered by verification. ISO 14696,
which provides guidance on the application of ISO
13485, states that a cleaning process needs consid-
eration of use and the controls in place to determine
whether some or all of the elements of validation are
required (5). Therefore, a decision to conduct a clean-
ing process validation is dependent on the outputs
of the cleaning process and the controls in place. For
example, Figure 1 shows a manufacturing process
flow that has an intermediate cleaning step. The sole
output of this intermediate clean step may be that the
parts are visibly clean. In this instance, verification
may be sufficient with no requirement for process
validation. On the other hand, the manufacturer
taking a risk-based approach has specified a level of
acceptable residues after intermediate cleaning. In
this instance, verification would not be sufficient and
process validation would be necessary.
In the above manufacturing process flow (Figure
1), the final cleaning step is the more critical cleaning
step of the two, as this is the last step in the process
to make sure that the medical devices are sufficiently
clean prior to packaging. The cleaning validation ap-
proach described within this paper is more applicable
to this final clean step. It consists of a number of
logical steps from identifying the risks to establishing
limits and from validation to process monitoring.
revIew PrOCeSS FlOw AND IDeNtIFy the MANU-
FACtUrINg MAterIAlS
In order to determine the cleanliness limits for manu-
facturing agents after the final cleaning process, it is
imperative to first evaluate the complete manufactur-
ing process. An overall process flowchart should be
created to demonstrate that the process has been ade-
quately assessed, and it should contain information on
the manufacturing materials that come in contact with
the product at each process step. Examples of manu-
facturing materials that must be considered include:
lubricants, detergents, wipes used during inspection,
and polishing agents. This manufacturing material
maybe attributable to one of the following groups:
Sebastian Clerkin
An organic residual; these are mostly insoluble in
water and include greases and oils
An inorganic residual; these are water soluble,
and examples include metal ions
Particulate; an example would be metallic par-
ticles left over from a cutting process.
Hazardous components of manufacturing materials
can be obtained from the Material Safety Data Sheet
(MSDS). The MSDS should be available for all the
manufacturing materials used in the process.
It must be noted, however, that the MSDS gener-
ally only lists the main components that are pres-
ent in a mixture. For example, the United States
Department of Occupational Safety and Health
Administration (OSHA) only requires that hazard-
ous constituents in excess of 1% be disclosed on a
MSDS (6). For carcinogens it is 0.1%. This is due to
the fact that MSDS are designed to protect the work-
ers, not identify potential hazardous contaminants
on a medical device.
There is also a risk that multiple constituents
below the 1% disclosure threshold could have a cu-
mulative effect on the intended use of the device.
Even though the MSDS does have these shortcom-
ings, it is still a very useful initial tool in identifying
potentially hazardous manufacturing agents.
When dealing with complex manufacturing
materials such as cutting fluids, it is a considerable
challenge to identify the individual components
as they contain many different chemicals. These
chemicals may not be identified by the supplier or
may be considered proprietary. In these instances,
it is imperative to liaise directly with the manufac-
turing material supplier to identify any potential
contaminants that could impact the intended use of
the device.
In addition, any process steps completed by an ex-
ternal supplier must also be evaluated. For example,
manufacturing material may be present on a supplied
component. These must be considered. This is why it
is crucial that the medical device manufacturer has a
written agreement in place with their supplier that no
changes are made to their manufacturing materials
without prior agreement.
Only after the overall process flow has been com-
pletely reviewed and all the potential contaminants
on the device identified can the manufacturer start
to consider their impact. Their impact is assessed by
conducting a risk analysis.
Special edition: Cleaning Validation 73
Sebastian Clerkin

Process Manufacturing Potential Potential Potential S L R Mitigation S L R

Step Material Hazards Effect Cause

Passivation Passivation Sodium Car- Residual 5 2 10 Use a passiv- 5 1 5

solution dichromate cinogen; Sodium ation solution
(CAS#: Irritant; dichromate that contains
7789-12-0) Mutagen present on no Sodium
the finished dichromate
device

Grinding Grinding wheel Metallic Third Metallic 4 2 8 Establish a 4 1 4

particulates body wear particulates particulate
of bearing present on specification
surface finished for finished
device device
table 1: Hazard Analysis. Severity (S) is scored1 to 5, 5 being the most severe; Likelihood is scored 1 to 5, 5 being the most likely; Risk index (R) is cal-
culated by multiplying the Severity score by the Likelihood score.

risk Analysis and Identification of Materials
of Concern
For the risk analysis, the impact of the contaminants
from a hazardous perspective and from an intended
functionality perspective must be considered.
A useful tool in identifying which contaminants
are of the most concern is to use a hazard analysis
and to ask the following questions:
Will too much of this contaminant be harmful to
the patient?
Will too much of this contaminant impact the
proper functioning of the device?
An example of a hazard analysis is shown in Table
I; the process steps, manufacturing material and par-
ticular agents are listed, and then the risk is consid-
ered. The mitigation from the hazard analysis can be
used to establish a particular cleaning limit or use of
an alternative manufacturing material.
eStAblIShINg CleANINg lIMItS
Now that the potential hazardous contaminants have
been identified, the acceptable level of contamination
on the medical device must be determined. These
levels or limits must be documented and scientifically
justified by the medical device manufacturer.
For toxic contaminants where there is known
toxicity data, ISO 10993-17 is very useful. It de-
scribes a method to determine the acceptable levels
of leachable material from a medical device using the
No Observed Adverse Effect Level (NOAEL) (7). The
NOAEL is the highest concentration of a material that
causes no significant adverse effects in the exposed
population. The standard takes this value and uses
it to calculate the tolerable intake (TI) for a specific
leachable substance. This approach can be used to
calculate limits for the pre-identified manufacturing
materials.
However, in many instances, the NOAEL is not
known, and so the manufacturer must rely on using the
LD50 values. LD50 is the median lethal dose. In other
words, the amount of a particular toxin that will kill 50%
of the population over specified time duration. These
LD50 values can be readily obtained from the MSDS.
The LD50 values are then used to calculate the Accept-
able Daily intake (ADI) using the following equation:
ADI = LD50 x mB/CF
Where:
LD50 = median lethal dose
mB = is the body mass of the patient population and is
generally defaulted to 70kg
CF= conversion factor
The conversion Factor (CF) is typically a factor be-
tween 100 and a 1000 and is derived to incorporate
uncertainty factors (UF) such as:
Extrapolation from animal to human tolerances
(typically defaulted to a factor of 10)
Inter-human variability (typically defaulted to a
factor of 10)
Additional UFs can be based on the type of
medical device (i.e., medical device class) and the
duration of exposure.
The weighting of each UF should be documented
and justified (8). The UFs are then used to calculate
CF:
CF = UF1 x UF2 x UF3

74 Special edition: Cleaning Validation


In most cases, a conversion factor between 100 and
1,000 is sufficient; however, there may be instances
where significant risks have been identified and a CF as
high as 10,000 may be appropriate (9). Refer to Kramer
et al.for further information on conversion factors (10).
Consider a real world example of the above ap-
proach using an alkaline cleaner:
The CF has been established as 1,000 to account
for no human toxicity data (factor of 10), inter-human
variability (factor of 10), and a short exposure time of
the device (factor of 10).
The LD50of the alkaline cleaner is 365 mg/kgrat.
The average human body weight is 70 kg.
This would give:
ADI/device = 365 mg/kg x 70 kg/1000 =25.55 mg/
device
Therefore, the cleanliness limit for this chemical
would be 25.55 mg per device.
Using this approach, a cleanliness limit can be
calculated for each specific toxin that was identified
during the risk analysis.
Obviously, this approach only identifies a cleanli-
ness limit for known toxins. It is not suitable for
calculating the cleanliness limit where there is a lack
of toxicological data available or the contaminants
have no associated toxicity but will impact the proper
functioning of the device.
For potential toxins where there is no readily avail-
able toxicological data, a series of spiking studies can
be completed. This is where the device is artificially
contaminated with known amounts of the potential
toxin. Biocompatible studies can then be completed
to determine the point of failure. The suite of ISO-
10993 standards provide a wealth of information that
can be used to define the biocompatibility studies
needed in establishing the failure point. Cytotoxic-
ity, sensitization, systemic toxicity, and genotoxicity
studies are examples of biocompatibility studies that
could be considered. The established failure point
can then be used to derive the cleanliness limit.
Another approach to spiking studies is to approach
the issue from the opposite end. In other words, in-
stead of finding the failure point, the device is spiked
with a known amount of the contaminant that is
above the level expected to be observed after clean-
ing. If this higher level is established as safe for the
patient, it can be defined as the cleanliness limit.
Spiking studies can also be useful if the risk analysis
has identified a potential cumulative effect of various
contaminants. In other words, if each contaminant is
treated independently of each other, a cleanliness limit
may be established that does not take into account a
potential cumulative effect. In this instance, the patient
may be exposed to unacceptable risk.
Spiking studies should also be considered for
Sebastian Clerkin
contaminants where the LD50 value is known but
the manufacturer has identified additional risks.
For example, if a carcinogen has been identified as
a potential contaminant, a spiking study may be
instigated with a genotoxicity and/or carcinogenicity
study as the endpoint.
Working out the cleanliness limits for non-toxic
contaminants, which have the potential to interfere
with the proper functioning of the device, needs to be
established by reviewing historical data or by spiking
experiments. The spiking study is conducted in a
similar manner as above, but instead of a biocompati-
bility study being the endpoint, the levels of contami-
nation are now evaluated against a specific functional
requirement for the device. For example, for a device
that has a bearing surface and device failure attribut-
ed to particulate contamination, a spiking study may
be developed to determine the level of particulate that
will induce the failure.
eStAblIShINg CleANINg teSt MethOD
Now that the cleaning limits have been identified
for specific manufacturing agents, the next step is to
decide on a method to quantify the levels. There are
two types of tests that can be developed:
A specific analytical test can be developed to
quantify a contaminant (refer to Table II for
examples).
A non-specific test can be developed to quantify
many different contaminants at the same time
(refer to Table II for examples).
There are pros and cons for both these approaches.
With respect to a specific test method, an accurate
measurement of a particular residue can be evaluated.
This can be very important when this residue has
been identified as being highly hazardous. However,
these specific methods are more difficult to imple-
ment and are more expensive; therefore, they are re-
ally only used when a specific risk has been identified
during the risk analysis.
Non-specific methods are more commonly used in
validating cleaning lines; they are less expensive and
easier to develop. However, due to their non-specific
nature, they do not give an accurate quantification
of any individual contamination, only a total level
of a group of contaminants. Generally, this can be
sufficient where the requirement is to demonstrate a
certain level of overall cleanliness.
When developing any test method, the following
factors should be considered:
Detection Limit; for example, the test method
must be sensitive enough to detect relevant levels
of the contaminants.
Special edition: Cleaning Validation 75
Sebastian Clerkin

Specific/Non- Test Method Description

Specific
Specific Infrared Spectroscopy A spectroscopic technique. Useful to identify and quantify
specific molecules
Grinding wheel Metallic particulates
Specific Grinding wheel Metallic particulates
Gas Liquid Partition Chro- Useful for quantifying a specific contaminant that can be
matography (GLPC) vaporized without composition
Specific High-Performance Liquid A method that separates a mixture allowing individual
Chromatography (HPLC) components to be quantified
Non-Specific Conductivity Measure of ionic compounds
Non-Specific Total Organic Carbon Quantitative measure of carbon contained within an or-
(TOC) ganic compound. A very good method of showing overall
cleanliness.
Non-Specific Visual Visual Assessment of cleanliness
Non-Specific Gravimetric Analysis Quantitative measure of mass of a solid within a solution
table 2: List of Test Methods. Each could potentially be used to assess outputs of a cleaning process.

Percentage recovery; the amount of contaminants
that can be recovered from the device must be
determined.
Reproducibility and Repeatability
Linearity
Specificity.
Once the test methods have been developed, they
must be qualified prior to being used in a cleaning
process validation. The test method validation must
demonstrate that the analytical method and the extrac-
tion and/or sampling method is repeatable. With re-
spect to the extraction method, it must be demonstrat-
ed that the contaminant can be consistently recovered
from the medical device. There is no point in having
a repeatable analytical method if the contaminant of
interest cannot be consistently extracted off the device.
Cleaning equipment Qualification (IQ)
The cleaning equipment must be qualified prior to
commencing the process validation. This will dem-
onstrate that the equipment is installed correctly and
functions as intended.
Operational Qualification (OQ)Challenge Condi-
tions
As part of the OQ phase, the critical process inputs
should be identified. It must be established how these
process inputs impact the cleanliness outputs. Brain-
storming and tools such as fishbone diagrams can be
very useful in identifying the critical process inputs.
Some examples of potential process inputs that could
be considered during a brainstorming are listed in
Table III.
To adequately challenge a cleaning process during OQ,
the worst-case product should be used. For medical
device manufacturers that clean multiple different
devices, identification of the worst-case product can
be a challenge. To aid in this identification, a worst-
case product matrix can be used to help determine the
worst-case product. Each product is scored according
to predefined criteria. These predefined criteria would
have a potential impact on the cleaning ability of the
process. Examples include the following:
Device geometry; more complex geometry is
potentially more difficult to clean
Surface area; greater surface area could be a
greater cleaning challenge.
Up-stream process flow; this could have an
impact on the cleanliness levels of the parts pre-
cleaning. A greater level of contamination on
the parts pre-clean would obviously be a greater
challenge to the process.
The scores against these criteria can then be tabu-
lated in the form of a matrix and used to calculate a
total risk score. See Table IV for an example.
Once one has established the critical process
inputs, these should be challenged within the OQ to
prove that product can be cleaned that meets the pre-
determined cleaning specifications under all antici-
pated manufacturing conditions.
PerFOrMANCe QUAlIFICAtION
PQ means establishing by objective evidence that the pro-
cess, under anticipated conditions, consistently produces
a product that meets all predetermined requirements (11).

76 Special edition: Cleaning Validation

 Sebastian Clerkin

Cleaning Process Inputs Comment

Tank Temperature Higher temperatures are generally more effective while lower temperatures are less
effective.
Cleaning Time Longer times are generally more effective while lower times are less effective.
Concentration Usually the higher the concentration of cleaning agent the better; however, at
higher concentrations there may be higher level of cleaning residual present on the
parts post-cleaning.
Cleaning Solution As parts are processed through the cleaning solution, it will become contaminated.
Contamination There is a risk that as the cleaning tank becomes dirtier, its cleaning effectivity will
decrease. To assess this, the change-out frequency of the tank, the level of con-
tamination on the parts, and the number of parts processed between change-outs
must be considered.
Flow Rate of Cleaning Higher flow rates are generally more effective while lower flow rates are less effec-
Agent tive.
Agitation Agitation should improve the cleaning effectiveness.
Ultrasonic Ultrasonic should improve the cleaning effectiveness.
Load A higher number of parts or dirtier parts may represent a greater challenge to the
cleaning process.
Rinse Parameters Rinse times, temperatures and volume are important in removing the cleaning
agent from the product.
Drying Parameters Time, temperature and airflow are critical parameters that can impact the dryness
of the parts. The geometry of some parts, such as the internal diameter of a narrow
tube, may make them difficult to dry. Another consideration is the temperature
used for drying. Too high a temperature could have an impact on the material
properties of the product.
table 3: Cleaning Process Inputs.

Worse Case Product Matrix

Product Number Surface Area Device Geometry Up-Stream Pro- Total Risk Score
cess Flow
1001 10 10 5 500
1002 7 5 10 350
table 4: Worst-Case Product Matrix. The product is scored between 1 and 10 for each criterion. These values are then multiplied together to give the
total risk score. The product with the highest risk score is then considered the worse case product.

To establish this, the medical device manufacturer must PrOCeSS MONItOrINg

demonstrate that the process in anticipated manufactur-
ing conditions is stable and can consistently produce
product to the predefined cleaning specifications. The
manufacturer can use tools such as control charts to
demonstrate that the process is stable as well as capability
analysis to show that the process is consistent.
With respect to identifying the anticipated con-
ditions of a cleaning process, factors that could be
considered are the following:
Material lots, for example, batches of cleaning agents
Environmental conditions
Multiple Shifts
Operators
Process operating windows defined in the OQ;
for examples, see above.
After the cleaning process is validated, it is desir-
able to implement process monitoring of key process
parameters and product outputs to maintain a state of
control. The action levels can be established during
the process validation.
ISO 14969 specifically states that the process pa-
rameters used for cleaning should be routinely moni-
tored in accordance with documented procedures
(12). Examples of critical process inputs that could be
monitored would be conductivity on a rinse tank or
concentration of cleaning agent in a cleaning tank.
The product outputs that would be monitored are
risk-based and would relate to the cleaning specifica-
tions established previously. For example, the medical
device manufacturer may consider monitoring the TOC
of the product on a regular basis to maintain confidence

Special edition: Cleaning Validation 77

Sebastian Clerkin
that the cleaning process is still in control. For newly de-
veloped cleaning processes, this monitoring can initially
be at a higher frequency and reduced as the manufac-
turer gains more confidence in their process.
revAlIDAtION
Obviously, changes to the cleaning system and process
should be assessed for their impact to the validation. In
addition, any changes to the manufacturing materi-
als used in the up-stream manufacturing processes
may have an impact on the cleaning validation. This
includes manufacturing steps completed by an external
vendor. If the change is deemed to have an impact, the
cleaning validation should be either repeated in part or
in full. Again, this needs to be a risk-based decision.
The addition of new products to the clean line should
be assessed to determine if they are a new worst-case.
If they are considered a new worst-case product, the
cleaning validation should be repeated. A recommended
approach to determining if revalidation is required with
a new product is to update the worst-case product ma-
trix described earlier with the new product. If the new
addition scores higher than the original worst-case, then
the cleaning validation must be repeated.
CONClUSIONS
The main steps in conducting a cleaning validation is
to assess the manufacturing material for each manu-
Figure 2: Overall Process Flow to a Cleaning Validation.
78 Special edition: Cleaning Validation
facturing process step, establish cleanliness limits,
and then validate the cleanliness test methods and
the cleaning process itself (Figure 2).
Cleaning validation does not need to be difficult.
If medical device manufacturers take a methodical
approach and base each decision on sound scientific
rational, they will be able to establish a cleaning
process that will consistently provide clean medical
devices to the market.
reFereNCeS
1. Code of Federal Regulations, Title 21, Quality System Regula-
tions, Part 820.70(e), 2013
2. Code of Federal Regulations, Title 21, Quality System Regula-
tions, Part 820.70(h), 2013
3. ISO 13485:2003, Medical devices -- Quality management sys-
tems -- Requirements for regulatory purposes, section 7.5.1.2.1.
4. Global Harmonisation Task Force Study Group 3, Quality
Management Systems Process Validation Guidance, January 2004
5. ISO 14969:2004, Medical devices -- Quality management
systems -- Guidance on the application of ISO 13485: 2003, section
7.5.2.1.1.5.
6. Code of Federal Regulations, Title 29, Part 1910 - Occupational
Safety and Health Standards, Subpart Z Toxic and Hazardous
Substances, 2013.
7. ISO 10993-17:2009, Biological evaluation of medical devices
-- Part 17: Establishment of allowable limits for leachable sub-
stances.
8. ISO 10993-17:2009, Biological evaluation of medical devices
-- Part 17: Establishment of allowable limits for leachable sub-
stances.
9. ISO 10993-17:2009, Biological evaluation of medical devices
-- Part 17: Establishment of allowable limits for leachable sub-
stances.
10. H.J. Kramer, W.A Van Den Ham, W. Slob, and M.N. Pieters,
Conversion Factors Estimating Indicative Chronic No-Ob-
served-Adverse-Effect Levels from Short-Term Toxicity Data,
Regulatory Toxicology Pharmacology 23, 249-255, 1996.
11. Global Harmonisation Task Force, Quality Management Sys-
tems - Process Validation Guidance Edition 2, January 2004
12. ISO 14969:2004, Medical devices -- Quality management sys-
tems -- Guidance on the application of ISO 13485: 2003, Section
7.5.2.1.1.6.
AbOUt the AUthOr
Sebastian Clerkin, Ph.D., B.Sc., is the Founder of GMP Advisory
Services Ltd. and has more than 10 years experience in labora-
tory, pharmaceutical, and medical device manufacturing environ-
ments. He currently provides validation and regulatory expertise
to the medical device industry. He has direct experience across a
broad range of medical device types and process technologies. He
has a keen understanding of FDA regulatory requirements, and
has a strong background in process development and validation.
He has a Ph.D. from the University of Bristol, which involved
studying the toxicological and biological effects of prosthetic im-
plants. He holds a B.Sc. in Biochemistry from University College
Dublin, and is a member of AAMI and ASTM. E-mail: gmpadviso-
ryservices@gmail.com
 Tim Sandle

Ensuring Sterility: Autoclaves,

Wet Loads, and Sterility
Failures | IVT
Tim Sandle, Ph.D.

ABSTRACT
Steam sterilization is a critical process in the pharmaceutical and
related industries. Modern autoclaves are computer-controlled and
reliably provide a defined sterilization cycle. When steam enters the
autoclave chamber and contacts with the item to be sterilized, steam
collapses (condenses). Water formed must be discharged through
condensate management or re-vaporized in order to prevent wet loads.
Repeated occurrences of wet loads are indicative of a major fault with
the sterilizer, potential non-sterilized materials, and other problems.
This paper considers some of the potential causes for wet loads and ad-
dresses some of the measures that can be taken to address occurrences.
Topics discussed include reasons for wet loads, causes including wet
steam, inadequate condensate removal, steam trams, pressure control,
and other causes; diagnosing problems by information collection, and
corrective actions. Corrective actions may include vacuum drying, heat-
ing the load before steam introduction, an air in-bleed phase, and other
approaches. Problems identified may be caused by a combination of
factors requiring a multidisciplinary team to evaluate potential causes.

INTRODUCTION
The most widely used sterilization method in the pharmaceutical in-
dustry remains steam sterilization in autoclaves (moist heat in the form
of saturated steam under pressure). This method is primarily appli-
cable to the terminal sterilization of products, stainless steel items, and
equipment not intended for single-use. With this method, sterilization
occurs as the latent heat of condensation is transferred to the load caus-
ing it to heat rapidly (1).
Modern autoclaves are computer-controlled and are generally very re-
liable. The autoclave acts as a pressure-cooker: Water boils at 100C at
atmospheric pressure; water boils at lower temperatures at lower pres-
sures, and water boils at higher temperatures at higher pressures. At a
steam over-pressure of one bar (a non-SI unit of pressure, exactly equal
to 100,000 Pascals), water boils at approximately 121C. This allows
the autoclave to produce temperatures above those that can ordinarily
be achieved. For sufficient time and with the correct conditions, such
temperatures can destroy bacterial endospores (2). More importantly,
the required temperature must be maintained for the required time in
order for the sterilization cycle to be successful (3).
Retrieving sterilized items from autoclaves only to discover that they
are wet results in a need to repeat the autoclave cycle. A wet item is
evidence of non-sterility. This is time consuming and expensive. This
forms part of the general rule with packages removed from autoclaves:
Any item that has been sterilized should not be used after the expira-
tion date has been exceeded or if the sterilized package is wet, torn, or
punctured (4).

Special edition: Cleaning Validation 79

Tim Sandle
When steam enters the autoclave chamber and
contacts with the product, it is important that the
steam collapses (condenses) on the product. This is in
order for the heat to be released to the load. However,
the formation of water must be discharged through
condensate management or re-vaporized in order to
prevent contamination of the product. Removal of the
excess water is important to prevent insulation of the
load from the steam.
Repeated occurrences of wet loads are indicative
of a major fault with the sterilizer. Water or damp
spots on the load insulate the intended product from
achieving temperature. This means that the load has
not been subjected to the required lethality and it is
therefore not sterile. During sterilization, the wetness
in the steam clogs the pores of packed loads and pre-
vents the steam from properly penetrating wrapped
loads or sealed pouches.
Furthermore, there is a post-contamination risk.
If the instruments or products absorb too much
humidity, resulting in wet loads at process termina-
tion, this dampness is an optimal habitat for bacteria
to thrive. Sterile items that become wet are consid-
ered contaminated because moisture brings with it
microorganisms from the air and surfaces. Moreover,
if the sterile barrier system is still wet, it has lost its
bacterial barrier properties and there is a major risk
of contamination.
In addition to issues of sterility, wetness can cause
corrosion or spotting on the instrument being steril-
ized. This can cause irreparable damage.
Wet loads present a problem of non-sterility; a
problem in terms of recontamination of a load; and
damage can occur to equipment. This paper consid-
ers some of the potential causes for wet loads and
addresses some of the measures that can be taken to
address the occurrence of wet loads.
REASONS FOR WET LOADS
Water is heated up to the boiling point to generate
steam. As water reaches its boiling point at approxi-
mately 100C, the temperature will no longer rise;
any energy added to the water will lead to evapora-
tion of water thus creating steam. If steam touches
a material or gas with a lower temperature than the
temperature of the steam itself, the steam will give
off energy and will condense and water is formed. In
order to be able to evaporate the condensate energy is
needed. The way to obtain sufficient energy is from
its environment.
The reason why wet loads occur is that the con-
densate becomes separated from the condensation
energy and therefore cannot re-evaporate. To avoid
wet loads, autoclave cycles must ensure that the
condensate remains in contact with the materials it
condensed on, or, alternatively, the process ensures
80 Special edition: Cleaning Validation
that all condensate that gets separated from its energy
finds its way to the drain. Thus it is critical that the
condensate made by each load item (including load-
ing furniture and wraps) must not migrate to other
objects.
There are three possible scenarios for wet loads.
These are:
Visible moisture on outside of packs
Moisture inside pack (e.g. moist towel)
Visible water inside the tray containing the load.
Each of these is of equal seriousness.
POTENTIAL CAUSES OF WET LOADS
In examining the causes of wet loads there are several
areas to consider. These are discussed below:
Steam Supply
Wet steam can also be associated with wet loads;
therefore steam needs to have a certain level pf dry-
ness (5). The moisture content of the steam (dryness
fraction) is measured as the weight of dry steam pres-
ent in a mixture of dry saturated steam and entrained
water. Poor steam quality (insufficient dryness) can
lead to excessive condensate formation within the
autoclave. This can arise when excessive demands are
placed on the steam supply (essentially the boiler).
Wet steam is steam at saturation temperature con-
taining more than 5% water (6). Wet steam lowers the
heat transfer efficiency of steam, which results in an
inefficient sterilization procedure.
Steam quality can be assessed by measuring the
condensate discharge just ahead of the sterilizer. The
measurement of steam quality is known as the dry-
ness fraction and this is dependent upon the steam
flow rate (7) where
= dryness fraction
ww = mass of water flow (kg, or lb / unit time)
ws = mass of steam (kg or lb / unit time).
For example, if the water content of the steam is
5% by mass, then the steam is said to be 95% dry and
has a dryness fraction of 0.95. In this example, the
steam quality would be considered unsatisfactory. A
satisfactory dryness fraction is generally regarded to
be 0.97 (8).
Non-condensable gasses can also affect steam qual-
ity. Noncondensable gases should be removed before
the steam leaves the boiler. When they are not, they
modify the steam from being pure water vapor to a
mixture of steam and gas. These become an unwant-
ed contaminant that does not allow steam to make

contact with the item. Further, there is the issue of
superheated steam (discussed below),
Areas of Condensate Formation
Insufficient condensate being removed from the dis-
tribution system can result in in water lying around
in the system and being drawn into the sterilizer
when the cycle demands it (pulsing). Signs of con-
densation can sometimes be seen from an examina-
tion of the inside of the autoclave chamber. This may
resemble water lines or circles of water droplets (9).
When this occurs, the steam circuit and supply
should be examined to determine where condensate
formation might occur. As steam travels, there are
many places and reasons why steam cools down and
condenses. Reasons include dead legs in pipework
or improperly trapped or insulated piping. When
steam comes into contact with piping connections it
can condense; moreover, poorly insolated piping will
cause steam to condense. Some condensate is natural
and is taken care of by steam traps which are placed
throughout the piping system. This can also be due
to inadequate maintenance of the system and steam
traps (see below).
The further away the steam line is from the heating
source/medium, then the more likely condensate is to
form. This is connected to insulation, for the further
away the steam line is then the greater the need for
insulation.
Other Issues with Pipes and Steam Supply
There are other factors relating to pipework and the
supply of steam, which may need to be considered.
These are:
Where steam pipes are not properly insulated
steam will condense in the pipe
The water separator should be close to the auto-
clave
If pipes run down towards the sterilizer, this can
cause steam to condense
The steam generator should be located close to
autoclave, minimizing pipework length
If a steam generator does not have sufficient
capacity for the autoclave, pressure drop may
occur at times of peak demand for steam and a
carry over of water can happen
If chemicals are added during the steam produc-
tion, operational issues may occur.
Steam Traps
Steam traps are intended to discharge condensate
while not allowing the escape of live steam. Problems
can arise if the steam trap is not functioning cor-
rectly. To guard against this, the jacket steam trap
should be inspected, along with the valve at the drain
Tim Sandle
port, to ensure that they are clear and functioning
properly.
Too much steam at too fast a rate can result in
excessive water formation that might overwhelm or
swamp steam traps. Therefore steam traps need to
be properly sized during the autoclave design phase.
Pressure Control
Given that autoclaves destroy microorganisms by
direct steam contact at the required temperature
and pressure for a specified time, pressure control is
important. Poor pressure control can cause variations
in steam velocities. This often arises because mainte-
nance of the pressure-regulating valves (PRV) in the
system is not always reliable. PRVs can change the
condition of the steam and, in theory, can actually
dry the steam as it passes through the valve.
Autoclave Loads
What is being loaded into the autoclave can be a con-
tributing factor. One mistake that some organizations
make is to pack the chamber full of product or con-
sumables in order to meet demand. However, dense
packing is frequently a cause of excessive condensa-
tion. This cannot always be satisfactorily flashed off
by subsequent steam injections. Furthermore, large
quantities of materials and complex packaging can
make effective steam circulation a challenge.
It is also worth considering:
The assembly of instruments in the set. For
instance, are the instruments well distributed in
the set? Is there too much metal mass in the set?
High density items wrapped in absorbent mate-
rial can help to reduce condensate (10).
How the set was packaged. It is important to con-
sider if the wrapper is too large (traps condensate
inside set), whether the package was taped too
tightly (traps condensate inside package), the
size of peel pouches for load contents (too much
metal mass in the pouch). In addition, before
using wrapping material, it should be held at
room temperature (20oC to 25oC) and at a rela-
tive humidity of 30 to 60%.
Loading techniques. Consider reducing the metal
mass in the load and check whether linen items on
the top rack and metal items on the bottom rack.
Rigid containers. Containers should be spaced
about one inch apart from each other. Stacking
should not be performed unless the container
manufacturer gives specific information on this
process. Place containers beneath other items
since they produce condensate.
Sterilizers should never be overloaded. There
should be sufficient space between items to allow
for steam to permeate around the package.
Special edition: Cleaning Validation 81
Tim Sandle
Packaging Materials
Packaging materials, used to wrap the items to be
sterilized, are probably one of the most important
parts of the sterilization process. Packaging provides
a protective barrier for the sterile item and prevents
it from becoming re-contaminated. When used in
aseptic processing, the packaging should be of low
particle generation.
Packaging needs to be well designed and of suffi-
cient porosity so that steam can pass through packag-
ing. In addition, packaging should be water repellent
to provide a sterile field. Furthermore, the packag-
ing material must not change significantly during
sterilization or release any substances that might
interfere with the action of the steam.
Other Factors
There are other factors that are linked with wet loads,
although these are less common with a well-designed
system. These are:
Autoclaves are equipped with heated jackets to
assist in drying of the load and to ensure that
condensate does not form on the chamber walls
during the hold period. If the autoclave jacket
has a different temperature than the chamber,
wet loads may occur. To avoid this, larger steam
jackets contain more heat and assist in the drying
process.
The autoclave carriage can be a factor. A stainless
steel carriage shelf creates more condensate than
an equivalent aluminum carriage. To add to this,
the shape and size of the chamber can greatly
affect the outcome of the load.
The preheat process. Here the problems range
from poor steam penetration, superheat, dam-
aged sterile barrier systems and other problems.
When the autoclave doors have a different tem-
perature than the chamber or walls. Or where
the jacket temperature is not uniform or lower
than intended.
When the item being sterilized touches the wall
of the autoclave.
Stacking two trays for holding loads within the
autoclave. The lower tray does not have sufficient
energy to dry the condensation.
Items being loaded in wet (anything going in
wet will come out wet.) When the steam makes
contact with the wet instruments additional con-
densate and cause dripping on other instruments
that may not dry.
Overloading the autoclave. Large quantities of
hard goods or even complex packaging can make
proper steam circulation a challenge. Other
parameters that may influence drying are the
density of the wraps and the design of the set
82 Special edition: Cleaning Validation
The materials that make up the load, for exam-
ple heavy metal mass can be a cause of wet
packs (11). Further, some loads are erroneously
designed where they become water traps or they
are positioned in such a way the orientation leads
them to retain water.
Condense drains can become blocked. It is
important to clean these at least twice a year.
If they become saturated they will not take the
water out of the steam.
Sometimes the wet loads follow a pattern and
may occur if the autoclave has not been used for
a few days. Here the cause may be the build-up
of condensate in the supply pipework.
When steam pipework is of an incorrect diam-
eter.
If the drain valve is not closed, leading to drain
water being returned.
Consistency of operator control of the sterilizers
and loading patterns.
There are some actions that mask the wet load
issue rather than address it. One example is with the
use of tray liners. Tray-liners do not solve the problem
but disguises it. Tray-liners have a high absorption
capacity and are placed in the tray under the items to
be sterilized. This way the condense is absorbed and
dispersed; however, non-sterility remains an ever-
present risk.
DIAGNOSIS
Collecting meaningful information is important for
establishing why wet loads occur. Whenever wet
packs occur, some of the information that should be
collected includes:
Date of wet pack(s)
Time of day
Load configuration (number of trays)
Number and description of trays that were
reported as wet
Selected cycle time (gravity or dynamic air
removal)
Cycle temperature and exposure time
Packaging material used for set(s) that were wet
Type of containment device used (e.g. Mayo tray,
metal mesh basket)
Person(s) who prepared/wrapped sets
Inventory list for set (to consider set configura-
tion and weight of set)
Vacuum integrity
Vacuum performance
Temperature/pressure calibration
Chamber level
Chamber/jacket trap performance
Chamber valves (solenoid and check)

Jacket insulation integrity
Sterilizer exposure time
Sterilizer dry time.
Analyzing such data can help to establish patterns
and these can feed into the root cause analysis.
CORRECTIVE ACTIONS
In addition to running a diagnostic to address some or
all of the above areas, there are other actions that can
be considered in order to reduce the possibility of wet
loads occurring. These include:
Vacuum Drying
In order to dry the load after the steam sterilization
step, some users are tempted to employ excessively
long deep vacuums at the end of the cycle. Because the
chamber is heated via the jacketing and the fact that
water will flash off into steam at a lower temperature
under vacuum, this technique can be relatively suc-
cessful. However, it will lead to an unnecessarily long
cycle time, and a potentially a non-sterile load can still
result since water can collect in certain areas.
Reducing Condensate Formation
A strong preventative measure to prevent the forma-
tion of condensate is to ensure that the load itself is
brought up to a certain temperature prior to the in-
troduction of steam. This can be accomplished by es-
tablishing a load heat up phase at the beginning of
the cycle. The phase functions at the dictate of jacket
set point, chamber drain (or load thermocouple) set
point and a phase timer. The phase is not advanced
until the set points are achieved and the phase timer
elapses. From here it would then pass to a purge (or
pre-vacuum) condition.
Air-in Bleed Phase and the Problem of Superheated
Steam
An alternative approach is to add a heated air in-
bleed phase at the beginning of the sterilization cycle.
For this, a heat exchanger can be installed to the air
in-bleed assembly to enable a combination of vacuum
and air in-bleed pulses. These would bring the cham-
ber load up to temperature more quickly, reducing
the condensate formation at the beginning of the
cycle. While a heat exchanger is not absolutely neces-
sary, it can be useful for particularly large chambers
or dense loads.
As an alternative to the heat exchanger, increasing
the jacket temperature set point can be considered.
Care must be taken to ensure that this does not in-
terfere with maintaining the temperature uniformity
within the chamber during the sterilize dwell phase.
Merely increasing the jacket temperature will result
in a super-heating of the steam.
Tim Sandle
Superheated steam occurs when the temperature
of the steam is higher than its saturation pressure.
This usually happens when the pressure of the steam
drops as it moves through the steam supply system.
This dries the steam and once there is a reduction in
moisture, less energy is required to raise the tempera-
ture of the steam. An autoclave fed with superheated
steam will function like a dry heat sterilizer, in which
the killing of microorganisms is far less efficient than
the optimal saturated steam required for sterilization.
A further cause of superheated steam is if the re-
duction valve is too close to the autoclave. This valve
should be at least 10 meters away. This is because
where the distance is too short the temperature will
not be reduced. A further reason relates to steam
pressure, where this is too high. Although fundamen-
tal to the laws of physics, it should not be forgotten
that temperature and pressure are related to each
other. Here it follows that the higher the pressure
then the higher the temperature.
Charge Rate Control
Some autoclaves do not have a proportional control
valve. This provides the control system capability to
control the ramp up (charge) rate of steam injec-
tion during the cycle. If a chamber does not have the
means to control the rate that steam that is added to
the chamber, this can lead to flooding of the chamber
with steam. This, in turn, results in instant condensate
formation. If the steam injection can be controlled so
that it as it is ramped up more slowly, the amount of
steam (and subsequent condensate) can be more easily
controlled.
Filter Sterilization
Some autoclaves are fitted with an air in-bleed filter
casing that can be sterilized in situ. Some manufacturers
of autoclaves will sterilize this filter during each cycle,
whereas other types require a special cycle to do this.
This second approach reduces the wear on the filter ele-
ment, resulting in a much longer life-time and prevents
repeatedly saturating the filter with steam. This latter
factor would result in excessive moisture being brought
into the chamber at the completion of the cycle. Thus
avoiding the in situ sterilization of the filter can be effec-
tive in avoiding excessive moisture.
CONCLUSION
This paper has addressed the issue of wet loads from
steam sterilization through an autoclave. Although
the probability of wet loads occurring can be avoided
through good validation and effective cycle develop-
ment, even autoclaves that have been in service for
years can be subject to wet loads. This paper has
outlined why wet loads are a major concern (non-
sterility, the risk of re-contamination and damage to
Special edition: Cleaning Validation 83
Tim Sandle
equipment) and it has presented some of the reasons
why wet loads can occur. The paper has also out-
lined, in addition to addressing the potential causes
of wet loads, some preventative measures that can be
adopted.
Humidity problems are a complex matter and are
in the majority of cases created by a combination
of different factors. Often wet load issues cannot be
solved immediately and time is required, together
with a multidisciplinary team, in order to address the
problem.
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ABOUT THE AUTHORS
Tim Sandle, Ph.D., Tim Sandle, Ph.D., is the head of the microbi-
ology department at Bio Products Laboratory Limited, a pharma-
ceutical organization owned by the UK Department of Health. Dr.
Sandle is, additionally, a visiting tutor at the School of Pharmacy,
Manchester University.