Validation Case Studies.

Paul L. Pluta ]
Cleaning Validation Failure
Unknown HPLC Peaks
Validation Case Study #4
Paul L. Pluta

Validation Case Studies discusses situations useful
to practitioners in validation. Each case presented
deals with a specific validation problem, elements of
which are described to demonstrate strategy to solve
validation problems. We intend this column to be a
useful resource for daily work applications.
Reader comments, questions, and suggestions are
needed to help us fulfill our objective for this column.
Case studies illustrating validation issues submitted
by readers are most welcome. Please send your com-
ments and suggestions to journal coordinating editor
Susan Haigney at shaigney@advanstar.com.
KEY POINTS DISCUSSED
The following key points are discussed:
A validation case study involving cleaning valida-
tion and the observation of unknown peaks in
high-performance liquid chromatography (HPLC)
chromatograms is discussed
Unknown peaks were observed from testing of swab
samples following cleaning of suspension products
containing insoluble active drug, suspending agents,
colors, and flavors
Equipment was visually clean
Following the original failures, a shotgun approach
with a new cleaning procedure was attempted but
was unsuccessful
Technical personnel then conducted laboratory
studies using multiple cleaning agents based
on the chemical and physical properties of the
product residues. A new cleaning procedure was
developed
Cleaning process parameters (i.e., concentration,
time, volume, etc.) were clearly specified in the new
cleaning procedure
Analytical scientists determined that the unknown
peaks were associated with the hydrophobic flavor-
ing ingredients in the formulation
An improved visual inspection procedure was
developed
The new cleaning procedure was successfully
validated
Post-validation monitoring confirmed the success
of the new procedure
Addition improvements in between lot cleaning
and campaign cleaning were implemented based
on knowledge gained in the investigation
The most important benefit was the demonstration
of a scientific and technical approach to problem
solving, cleaning method development, understand-
ing residue chemistry, specifying process parameters,
etc.a scientific and technical basis for validation
The site implemented a new approach to cleaning
method development including analytical support.

[
ABOUT THE AUTHOR
Paul L. Pluta, Ph.D., is a pharmaceutical scientist with extensive technical and management
For more Author e xperience in the pharmaceutical industry. He is editor-in-chief of the Journal of GXP Compliance
information, and the Journal of Validation Technology. He is also adjunct assistant professor at the University of
go to Illinois College of Pharmacy in Chicago, Illinois USA. He edited and contributed chapters to Cleaning
gxpandjvt.com/bios and Cleaning Validation, published by Davis Healthcare International in 2009. He may be reached at
paul.pluta@comcast.net.

g x p a n d j v t . co m Journal of Validation Technology [Autumn 2010] 65

Validation Case Studies.
INTRODUCTION
This case study was provided to the Journal of Validation
Technology by a reader who requested anonymity. The
circumstances describe an actual occurrence. This dis-
cussion will provide the following:
Background. A description of the event, the key
issues to be addressed, and applicable current good
manufacturing practice (CGMP) requirements
Investigation. Interviews and actions conducted
to investigate the event.
Discussion. Key information, activities, and
analysis.
Corrective action and preventive action
(CAPA). Actions and improvements implemented.
These included the cleaning procedure change, speci-
fication of process parameters, equipment inspec-
tion procedure change, training on new procedures,
cleaning validation, and associated activities.
Post CAPA. Verifying and maintaining validation
and performance.
Other activities. Improvements implemented as
result of knowledge gained and activities associated
with the above, including the site approach to clean-
ing validation including specific cleaning process
development practices.
COMPLIANCE EVENT BACKGROUND
A small molecule pharmaceutical company was complet-
ing an upgrade of its cleaning validation program. Clean-
ing validation for a family of legacy liquid suspension
products was initiated. The product family comprised
four different products having multiple dosage strengths
of the same active pharmaceutical ingredient (API). The
product formulations were essentially identical, but had
different colors and flavors. The API was a basic com-
pound that was insoluble in neutral and alkaline pH.
The cleaning method for all products comprised agi-
tated immersion cleaning of the mixing tank with water
(fill tank with water and run mixer for 30 minutes). All
associated equipment including the impact mill, homog-
enizer, transfer lines, and filling equipment were also
cleaned using only water. Cleaning was accomplished by
extensive soaking and flushing. Equipment was always
visually clean at the completion of cleaning. An HPLC
analytical method was developed to quantitate residual
active drug from swab samples. Because no cleaning
agent was used, testing for residual cleaning agent was
not necessary. Worst-case swab sampling locations were
identified on manufacturing equipment.
The first product lot was manufactured and cleaning
was completed. Equipment was visually clean. Swab
66 Journal of Validation Technology [Autumn 2010]
sampling was done. Cleaning validation analytical test
data indicated that no active drug was present in all swab
samples. However, several other unidentified peaks on
some of the mixing tank samples were observed in the
chromatograms. Some of these peaks were at significant
levels. A second product lot was simultaneously manu-
factured. Manufacturing was completed and cleaning
was done. The equipment was visually clean. Swab
sampling was done on the second lot. Cleaning valida-
tion analytical test data again indicated that no active
drug was present in all swab samples. However, several
unidentified peaks on some of the mixing tank samples
were again observed in the HPLC chromatograms. Near-
ly identical test results after cleaning both lots confirmed
that cleaning was not successful.
Management decided to restart the cleaning validation
using the best cleaning procedure available in the plant.
This cleaning procedure used an alkaline cleaning agent.
Another product lot was manufactured. Equipment was
cleaned using the new procedure with alkaline cleaning
agent. Equipment was visually clean after cleaning. Swab
sampling was done. Cleaning validation analytical test
data again indicated that no active drug was present in
all swab samples. Several other unknown peaks on some
of the mixing tank samples were again observed in the
chromatogram. The new cleaning procedure was unsuc-
cessful in completely cleaning manufacturing equipment.
What are the Issues?
There were several critical issues to be investigated
as follows:
Why was the equipment dirty?
The sampling sites that had the unknown peaks were
consistent between the three lots. Problem sites were
located on the tank wall and the mixing blade shaft
just above the product-air interface. Why were these
sites not adequately cleaned?
Did manufacturing personnel correctly perform the
cleaning procedure? Perhaps personnel consistently
did not adequately clean certain areas of the equip-
ment that were shown to be dirty?
Three people inspected the equipment and certified
that it was clean. Was the equipment properly inspect-
ed? Was the equipment dry when it was inspected?
Why wasnt the residue seen?
Is the cleaning procedure technically sound? Water
has been previously used to clean the equipment. The
latest trial used alkaline agent. Both were unsuccessful
What was the source of the unknown peaks? Did
the analytical people ever have experience with these
peaks or ever see these peaks during development?
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CGMP Requirements
Relevant good manufacturing practice (GMP) require-
ments applicable to the event are listed as follows:
Subpart DEquipment
211.67. Equipment Cleaning and Maintenance.
Subpart JRecords and Reports
211.180. General Requirements.
211.182. Equipment Cleaning and Use Log.
211.188. Batch Production and Control Records.
211.192. Production Record Review.
INVESTIGATION
Investigation and ultimate resolution of this event
required involvement of several groups. These
included personnel involved in the incident (i.e.,
operations people who cleaned the equipment
and quality assurance people who inspected the
equipment), technical personnel responsible for the
cleaning procedure, and laboratory personnel who
developed the analytical method and performed
the analysis of the cleaning samples. There were
many details that needed to be investigated or con-
firmed. Personnel from all groups were interviewed
to address the issues.
Manufacturing Personnel Interviews
Manufacturing personnel confirmed that they per-
formed cleaning as required by procedure. The mixing
tank did not require human involvement in clean-
ingall process steps were automated. All associated
equipment that was manually cleaned did not have
any unknown peaks. All equipment was visually
clean. The manufacturing supervisor verified that
the equipment was visually clean. Quality unit per-
sonnel who inspected the equipment also verified
that all equipment was visually clean. All inspec-
tions were conducted after the equipment was dry.
Quality personnel went back to the equipment after
the unknown peaks were observed and viewed the
equipment. All equipment still appeared visually
clean. This subsequent inspection was conducted
with additional light.
Technical Personnel Evaluation
Technical personnel had no previous experience with
the cleaning method. The cleaning method had been
established many years ago and never required techni-
cal evaluation. Technical personnel recommended
laboratory studies to evaluate available cleaning
agents, cleaning process parameters, and related fac-
tors in a systematic way.
g x p a n d j v t . co m
Paul L. Pluta.
Analytical Laboratory Personnel
Comments
Laboratory personnel who developed the analyti-
cal method for cleaning validation had not seen the
unknown peaks in previous chromatography. Labo-
ratory personnel recommended that all formulation
components (inactive ingredients including flavors and
colors) be tested to determine if other components were
associated with the unknown peaks.
DISCUSSION
Information obtained through interviews and subse-
quent experimental work enabled good understanding
of the problem and ultimate solution. Cleaning was
done according to procedure, and equipment was visually
clean. Cleaning procedure performance, any overlooked
steps, or other errors did not occur and could not have
caused the cleaning problem.
Technical Cleaning and the Cleaning
Procedure
Technical personnel conducted laboratory studies
using acidic, neutral, and alkaline cleaning agents.
These trials included studies on the active drug as well
as on the complete formulation. The active drug was a
basic compound and was the primary component in
the formulation. The acidic cleaning agent completely
dissolved the basic drug (an acid-base reaction). After
acid treatment, the product solution was clear and
colored consistent with the dye color in the formula-
tion. The neutral and alkaline cleaning agents did
not have this same effect and were much less effective
in cleaning. The product solution remained opaque
and colored. The API did not dissolve in the alkaline
cleaning liquid or in water. Dissolution of the API with
the acidic cleaning agent significantly improved the
cleaning process because all insoluble residues were
eliminated. The API residue was completely dissolved
and able to be easily rinsed from the manufactur-
ing equipment. Cleaning of the other formulation
components was best accomplished with the alkaline
cleaning agent. The concentrations of both the acidic
cleaning agent and the alkaline cleaning agent were
optimized in laboratory studies using the formula-
tion components.
A new cleaning procedure was developed in which
the manufacturing equipment was initially treated
with acidic cleaning agent. The cleaning agent solu-
tion, containing the dissolved alkaline drug residue,
was then rinsed and discarded. Cleaning with an
alkaline cleaning agent was then conducted. The
Journal of Validation Technology [Autumn 2010] 67
Validation Case Studies.
equipment was then rinsed and dried. This new
cleaning procedure was successfully transferred to
commercial scale equipment.
The volume of cleaning liquid in the tank was
exactly specified to exceed the volume of product
previously manufactured. This ensured that cleaning
agents manufacturing equipment surfaces far above
the product-air interface that had been the sites of
the swab sampling failures. Previous wording of the
cleaning procedure simply stated fill tank, which
might have contributed to inadequate cleaning at the
level of the product-air interface.
Analytical Method
Samples of all formulation ingredients including the
suspending agent, colors, and flavors were provided
to the lab. HPLC retention times were determined
for several formulation components in the API HPLC
procedure. Testing of all components using the HPLC
procedure for the active drug indicated that the flavor
components were the unknown peaks in the prob-
lem chromatograms. These results indicated that if
the cleaning procedure could be improved to elimi-
nate these components, cleaning could be success-
fully accomplished. Studies conducted with only the
flavor components indicated that the new cleaning
procedure (acid cleaning agent followed by alkaline
cleaning agent) would successfully clean the flavor
components from equipment surfaces.
Inspection Procedure
Laboratory studies with individual formulation ingredi-
ents demonstrated the difficultly in seeing the problem
residue on the equipment surface. The problem ingre-
dients were clear hydrophobic oils that remained on
the tank wall and mixing blade shaft but were nearly
invisible. A new equipment examination procedure was
developed in which the problem areas on the equip-
ment were carefully examined with additional light.
CAPA
The following CAPA and associated activities were
conducted:
New cleaning procedure. A new cleaning pro-
cedure comprising the following was implemented:
1. Acid cleaning agent treatment
2. Rinsing with potable water
3. Alkaline cleaning agent treatment
4. Rinsing with potable water
5. Rinsing with purified water
6. Drying.
68 Journal of Validation Technology [Autumn 2010]
 leaning process parameters such as exact liquid vol-
C
umes, concentrations of cleaning agents, rinsing times,
etc., were specified for all steps.
Cleaning procedure validation. The new clean-
ing procedure was validated. Cleaning validation
included swab sampling of most difficult-to-clean
locations on all equipment. Additional sampling of
locations that had previously demonstrated residual
formulation ingredients was also conducted. All test
data passes residue limit acceptance limits and there
were no unknown HPLC peaks in any chromato-
grams. Three lots were tested.
Training. All personnel were trained on the new
cleaning procedure.
Impact on past manufacturing. Previous lots
were not considered to be at risk of cross contamina-
tion because the unknown peaks were identified to
be flavor ingredients. The same flavor ingredients
were present in all formulations but differed in con-
centrations (there were different amounts and ratios
of flavors to provide different dominant flavors in
each product).
Inspection. The visual inspection procedure for
cleaned equipment of these products was enhanced.
The modified procedure included increased light
and specific examination of problem areas on the
mixing tank.
Visual inspection training. Manufacturing and
quality unit personnel were trained on the new
visual inspection procedure.
Documentation. All work associated with the
original validation failure including technical clean-
ing method development and analytical determina-
tion of unknown peaks was documented and filed
in the validation library.
POST CAPAVERIFYING AND MAINTAIN-
ING VALIDATION AND PERFORMANCE
Swab sampling and analytical testing of problem areas
on the manufacturing tanks was continued for three
additional lots to confirm the successful solution to the
cleaning problem. Only the equipment sites that had
previously exhibited unknown peaks were sampled and
tested. All test data from the three additional lots were
negative and no additional HPLC peaks were observed.
OTHER ACTIONS
The knowledge gained from understanding the prop-
erties of the product residue and development of the
acid and alkaline cleaning procedure was applied to the
between-lot rinsing procedure for these products. When
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campaigns of multiple product lots of this product fam-
ily were manufactured, extensive rinsing with water was
conducted between lots. This procedure was changed to
include rinsing with citric acid (also a formulation ingre-
dient) followed by rinsing with purified water. The acid
pH of the citric acid solution dissolved residual insoluble
active drug. This procedure provided easier, faster, and
more complete cleaning compared to the water rinsing
procedure. Eventually the new between lot procedure
and the new cleaning procedure were used to validate
cleaning after an extended manufacturing campaign
resulting in time and cost savings.
SITE APPROACH TO CLEANING
VALIDATION
This event ultimately resulted in a significantly improved
cleaning procedure for the suspension product group,
successful cleaning validation, and improved and more
efficient ongoing cleaning. Further benefit to the site
an undocumented benefitwas the demonstration of
a scientific and technical approach to problem solving,
cleaning method development, understanding residue
chemistry, specifying process parameters, and so on. The
shotgun approach to cleaning whereby the best clean- paign cleaning were also implemented as result of the
ing method at the site was unsuccessfully tried further
demonstrated that there should be a technical rationale
for such activities. One size fits all does not work in
pharmaceutical cleaning validation.
Analytical Testing Changes
Another improvement to the site approach to clean-
ing process development involved the support work by
the analytical lab. The analytical lab proactively tested
other formulation ingredients that might potentially
become unknown HPLC peaks in future unsuccessful
cleaning validation. Previous analytical work in support
of cleaning process development focused on only the
residual API. If unknown peaks were observed when
this new development approach was used, laboratory
data could be reviewed to determine the identity of these
g x p a n d j v t . co m
Paul L. Pluta.
peaks. This approach provided excellent background
information during cleaning procedure development
and problem solving.
CONCLUSIONS
A case study involving a cleaning validation failure
on a legacy product was described. The problem was
addressed through a collaborative effort involving
scientists from the product technical group and the
analytical laboratory. Their approach started with an
understanding of the chemical and physical proper-
ties of formulation ingredients. Once these properties
were understood, development of a new cleaning pro-
cedure with appropriate cleaning agents was accom-
plished through a systematic technical approach.
A new enhanced cleaning procedure was successfully
developed. Ultimately the new cleaning procedure
was easily validated without any concernsconfirm-
ing the scientific basis and thorough development
work that was the basis for the cleaning method.
A post-validation monitoring protocol was also complet-
ed to verify successful validation. Additional improve-
ments to the between-lot cleaning procedure and cam-
knowledge gained in the problem-solving exercise. Ana-
lytical testing provided excellent support to the problem
solving effort. A revised approach to cleaning process
development involving identification of potential con-
taminants evolved from the activities of this case study.
This case study demonstrated the benefit of a scientific
and technical approach as the basis for validation. JVT
ARTICLE ACRONYM LISTING
API Active Pharmaceutical Ingredient
CAPA Corrective Action and Preventive Action
CGMP Current Good Manufacturing Practice
GMP Good Manufacturing Practice
HPLC High-Performance Liquid Chromatography
Journal of Validation Technology [Autumn 2010] 69