DRY HEAT DEPYROGENATION

CONSIDERATIONS:
Design Requirements for Facilities and
Equipment and Their Qualification – A
Case Study

Patrick J. McCormick, Ph.D.

pmccormick@bausch.com
+ 1 585 338 8390
Photo courtesy of Texwipe
Pyrogens and Endotoxin
• Sterilization and
depyrogenation Endotoxin tied to sterilizers.
Hospital traces fatal
seldom drive the outbreak. Saturday,
development of December 16, 1995.
Cookson, S.T. et al. Pyrogenic
new products, Reactions in Patients Undergoing
Cardiac Catherization Associated with
but are essential Contaminated Glass Medicine Cups.
to the safety of Catherization and Cardiovascular
Diagnosis 42:12-28(1997)
our products and
the health of our Outbreak traced to enzyme detergent
contaminated with > 104 CFU/ml; 434
customers. EU/ml; reprocessed cups had 2,250 EU.
Pyrogens and Endotoxin
• Pyrogen: any substance that induces a fever.
• Exotoxin: soluble protein secreted by
microorganisms that is toxic to cells (botulism
toxin, diphtheria, toxic shock syndrome, etc.).
• Enterotoxin: soluble protein secreted by
microorganisms in the intestine (food
poisoning).
• Endotoxin: high MW complex associated with
the cell wall of G (-) bacteria that is pyrogenic
in humans and specifically interacts with LAL.
Pyrogens and Endotoxin
Endotoxin:
• Threshold pyrogenic response of 1 ng/kg where
5 EU is equivalent to 1 ng E. coli (EC-2) reference
endotoxin.
• Complex host response (inflammatory cytokines)
can lead to high fever, severe tissue damage and
death.
Depyrogenation:
• Validated process designed to remove or
inactivate endotoxin (ANSI/AAMI ST72:2002).
Pyrogens and Endotoxin
• For most medical devices
an endotoxin limit of 20
EU/device is generally
recognized, although some
medical devices such as
IOLs may have lower limits
(ISO 11979-8; 2 EU/IOL).
Consult applicable
regulatory guidelines (ISO,
EN, FDA).
Pyrogens and Endotoxin
• For most pharmaceutical
applications the generally
accepted endotoxin limit
varies with the route of
administration:
– 5 EU/kg/hr – intravenous;
– 0.2 EU/kg/hr – intrathecal.
• Consult pharmacopeias
and applicable regulatory
guidelines.
Dry Heat Sterilization - Kinetics

• Well defined 1st 1000000

1/2 cycle
100000
order inactivation window
10000
kinetics.
1000
• z = 20 oC. 100
• FH at 170 oC. 10
• Total kill of 106 1
0.1
spores of Bacillus 63%
0.01
atrophaeus at half 0.001
pos. 1%
cycle based on 0.0001 pos.
overkill approach of 0.00001 10-6
ISO 14937/14161. 0.000001 SAL
exposure
Dry Heat Depyrogenation - Kinetics
The successful validation of depyrogenation is generally
accepted as evidence of the successful validation of dry
heat sterilization due to the much greater resistance of
endotoxin to dry heat as compared to bacterial spores.

Dry heat exposure* Sterilization Depyrogenation

120 minutes 160 oC None
60 minutes 170 oC None
30 minutes 180 oC 250 oC

* Actual exposure time will vary with load and conditions.

Dry Heat Depyrogenation - Kinetics

• Endotoxin inactivation kinetics are 2nd order as

opposed to first order (sterilization).
– Tsuji,K. and Harrison, S.J. 1978. Appl. Environ. Microbio.
36(5):710-714.
– Akers. M.J. et al. 1982. JPDA 36(1):23-27.
• Empirical validation - a three log reduction of
endotoxin is generally accepted as evidence of
successful depyrogenation and sterilization.
Biological indicators (B. atrophaeus) typically are
not employed.
Dry Heat Depyrogenation - Equipment
Heat transfer:
• Conductive:
– Heat transfer via direct physical contact.
• Convective or radiant:
– Heat transfer via fluid medium (liquid or gas).
– Air flow/shadowing effects/HEPA filtration.
• Radiation:
– Heat transfer via electromagnetic radiation
(infrared or microwave).
Dry Heat Depyrogenation - Equipment
Batch sterilizer:
• Widely used in industry.
• Simple, rugged design.
• Fans circulate heated
air throughout load.
• Ideal for varying load
types, small loads, and
infrequent use.
PDA Technical Monograph #3
Dry Heat Depyrogenation - Equipment

Tunnel sterilizer:
• Operates continuously.
• Ideal for large load sizes.
• Fast processing time.
• Convection, infrared,
flame sterilizers.
• Variable heating zones.
• Airflow opposite product flow. PDA Technical Monograph #3
Dry Heat Depyrogenation – Facilities Considerations
• Integration / Process flow – How is the integration
of depyrogenation oven with other equipment such
as vial washers, filling and capping machines,
packaging equipment, etc. to be realized such that
uninterrupted processing may be achieved?
• Total process throughput is tied to rate limiting
step. Select oven and validate depyrogenation
cycle time appropriate to overall manufacturing
process.
Dry Heat Depyrogenation - Facilities Considerations
• Capacity – Does the oven have sufficient capacity to
meet current and future processing needs?
• Classification – Is the classification of the oven
compatible with the classification of the area where it
will located and the overall manufacturing process?
Most depyrogenation ovens are rated Class 100 and
should therefore be suitable for application in vial
and equipment prep areas of aseptic manufacturing
facilities.
Dry Heat Depyrogenation – Facilities Considerations
• HVAC / Utilities – Are current HVAC controls and
utilities (electrical, water, air) adequate to meet the
needs of the depyrogenation oven? How much
added stress will be placed on the existing HVAC
system and utility supply and what impact will this
have on the operation of other equipment ?
• Installation – Are the dimensions of the oven such
that it can be transported to and installed in the
desired location or will alterations to the facility and
existing layout be necessary?
Dry Heat Depyrogenation – Facilities Considerations

• Building Codes – Are there any particular

considerations with regard to local or regional
building codes such as the need for seismic
anchoring or electrical grounding requirements?
• Workplace – Is the unit quiet during operation or
will acoustic protection be necessary? What about
additional heat stress and vibration? How will this
affect production operators or other manufacturing
equipment?
Dry Heat Depyrogenation – Facilities Considerations

• Maintenance / Calibration / Validation – Does the

facility have adequate resources to support the
maintenance, calibration, and validation needs of
the oven or are additional staff and equipment
needed? How to plan for interventions?
• Change Control – Is the design of the oven such
that it can be readily relocated should the layout of
the facility be changed?
Dry Heat Depyrogenation – Equipment Considerations
• Control systems – State of the art. Compatible
with controls systems of other manufacturing
equipment? This may be a problem if your
washer and filling equipment is 20 + years old.
• Control systems – Operator interface and
datalogging capability. How will critical cycle
documentation be captured and stored? Accuracy
and precision of recorders? Backup? What about
EMF/RFI interference issues?
Dry Heat Depyrogenation – Equipment Considerations
• Pressure Balance – Is the airflow of the oven
such that the appropriate pressure balance can
be maintained so that product moves to
progressively “cleaner” zones and classification of
the surrounding areas is not impacted?
• Air pathways – Are there provisions for the
sterilization of air pathways within the oven,
particularly the cool down zone? How is air quality
of the area to be maintained during repair and
maintenance of the oven?
Dry Heat Depyrogenation – Equipment Considerations

• Materials of construction – 304L SS typical with

gauge and finish appropriate to application and
loading conditions.
• Door interlocks – Appropriate to product flow with a
safe locking sequence in the event of power loss
(prevent product from exiting chamber without
being adequately depyrogenated).
Dry Heat Depyrogenation – Equipment Considerations

• Alarms – Low/High temperature, airflow, belt

speed, emergency stop, etc.
• Contamination – Is the oven a potential source of
particulate (filter binder material, material shed
from moving parts, etc.) or chemical contaminants
(lubricants) during routine operation? Is the use of
DOP a concern during validation of HEPA filters?
Dry Heat Depyrogenation – Case Study
• Retisert™ intravitreal
drug implant for
treatment of chronic
non-infectious uveitis
affecting the posterior
segment of the eye.
• Uveitis is a leading
causes of blindness in
the Western world.
Dry Heat Depyrogenation – Case Study
Retisert implant:
• Manufactured by
Bausch & Lomb in
Waterford, Ireland.
• Depyrogenation
validation performed in
association with
Autocal Ireland Ltd and
Microchem Labs.
Dry Heat Depyrogenation – Case Study
Retisert implant:
• Manufactured under
cleanroom conditions.
• Terminally sterilized by
gamma irradiation
(minimum 25 kGy) as per
EMEA Decision Trees.
• Depyrogenation at 250 +
15 oC for NLT 60 minutes
of fixtures and tooling in
contact with product.
Dry Heat Depyrogenation – Case Study
• Depyrogenation strategy included as part of overall
Cleaning Validation Strategy and Assessment.
• Important to bridge development phase in order to
maintain continuity of approach.
• Assess each step of process to identify critical risk
areas for depyrogenation (FEMA/HACCP).
• The move to a rigorously controlled manufacturing
environment may decrease the need for extensive
depyrogenation as compared to the preceding
development phase.
Dry Heat Depyrogenation – Case Study
Oven considerations:
• Double door unit (Wash in / Wash out).
• Door lock sequencing including power failure.
• Size of depyrogenation oven was a major factor due
to existing space restrictions.
• Oven must interface with equipment prep area to
ensure effective HACCP and manufacturing flow.
• Must be compatible with existing HVAC/utilities.
• Must be consistent with area classification.
• Must allow access for maintenance and calibration.
Dry Heat Depyrogenation – Case Study

Gruenberg L55H8.3PTSS:
• Chamber interior:
– 20” x 30” x 24” (8.3 ft3/ 0.2 m3).
• Chamber exterior:
– 68” x 44” x 70”.
• Bio sealing flange.
• Horizontal flow of heated Class
100 HEPA filtered air.
• Heat exchanger (cold water) for
cool down.
http://www.epsovens.com
Dry Heat Depyrogenation – Case Study

Gruenberg L55H8.3PTSS:
• Temperature range:
– Max. 280 oC.
– Operating 250 oC.
• Temperature Control:
– 0.25% of scale at 250 oC.
– Ramp rate > 1.5 oC/min.
• Temperature uniformity:
– + 5 oC at 250 oC. http://www.thermalproductsolutions.com
Dry Heat Depyrogenation – Validation
• EP 5.1.1 General Texts on Sterility.
• USP <1211> Sterilization and Sterility Assurance.
• EMEA: Decision Trees for sterilization.
• ISO 14937 – General requirements for sterilization.
• PDA TR 3 – 1981 Dry Heat Processes for sterilization
and depyrogenation; revision 2008.
• PDA TR 7 – Depyrogenation.
• FDA Aseptic Processing Guidelines 2004.
• AAMI ST63 – 2002 Dry Heat sterilization.
• HTM 2010 and other regional requirements.
Dry Heat Depyrogenation - Validation

Documentation:
• Validation Master Plan.
• User Requirements Specification (URS).
• Request for Quote (RFQ).
• Proposal from vendor (including drawings).
• Functional / Design Specifications.
• Factory Acceptance Test (FAT).
• Validation Documentation (IQ/OQ/PQ).
Dry Heat Depyrogenation – Validation
General Validation Strategy:
• Factory Acceptance test (FAT).
• Installation Qualification (IQ).
• Operational Qualification (OQ) – 3 cycles.
• Cycle Development / Heat Penetration (HP) studies.
• Reduced Cycle testing – 1 cycle.
• Performance Qualification Testing (PQ) – 3 cycles.
• Annual revalidation – One ea. OQ and PQ cycle.
Dry Heat Depyrogenation – FAT
Factory Acceptance Test:
• Verify physical dimensions of unit.
• Verify loading system operates correctly.
• Verify access ports for particle testing.
• Conduct heat distribution test (empty chamber).
• Conduct load test.
• Verify proper ∆ P across HEPA filters.
• Personnel training in operation of unit.
Dry Heat Depyrogenation – IQ
Installation Qualification (IQ):
• “Obtaining and documenting evidence that
equipment has been provided and installed in
accordance with its specification.” ISO 14937:2002.
• Do we have all the parts?
• Will the unit fit into the allotted space?
• Do we have the necessary utilities?
• Is the unit to spec?
Dry Heat Depyrogenation – IQ

Confirm physical installation to specifications:

• Positioning of unit. • Ducting and plumbing.
• Leveling/anchoring. • HEPA filter installation
• Baffles and panels. and certification.
• Seals and gaskets. • Environmental and
• Insulation. Safety inspection.
• Utilities (air, water, • Manuals and drawings.
electrical, etc.). • Spare parts list.
Dry Heat Depyrogenation – IQ
Verify Calibration:
Oven Instrumentation: Calibration equipment:
• Temperature control. • Tachometer.
• Chart recorder. • Temperature calibrator.
• High limit alarm. • Thermocouple bath.
• Intake pressure. • Data logger.
• Exhaust pressure. • Pressure calibrator.
• Recirculation pressure. • Thermal anemometer.
• Chamber pressure. • Particle counter.
Dry Heat Depyrogenation – IQ
• Note: HEPA filters are fragile and should
not be shipped installed in the unit. New
filters should be put in place once the unit
has been received and “burned in” at the
maximum operating temperature as per the
manufacturer's recommendation. This can
create an odor and haze in the air. Do this
over the weekend to minimize disruption to
others.
Dry Heat Depyrogenation – OQ
Operational Qualification:
• “Process of obtaining and documenting evidence
that installed equipment operates within
predetermined limits when used in accordance with
its operational procedures.” ISO 14937: 2000.
• Does the unit perform as promised?
• Let’s take it for a test drive…
Dry Heat Depyrogenation – OQ
Verification of PLC software:
• Generate Control Software Qualification Report.
• Verify correct software version number.
• Verify electronic backup of same software version
provided on permanent magnetic media.
• Verify hard copy of PLC ladder/logic provided.
Dry Heat Depyrogenation – OQ
Operational Set-Up Checks:
• Verify speed controller set as per manual.
• Verify high-limit controller set as per manual.
• Verify chart recorder set as per manual.
• Verify circulation fan rotating in correct direction.
• Verify fan operating at correct rpm (tachometer).
• Test controls, alarms, indicators, safety devices, etc.
Dry Heat Depyrogenation – OQ
Empty chamber test: Avg. = 244.61 oC
• Place 12 TC’s as
indicated w/in 8 cm
of chamber surface.
• Oven set at 250 oC.
• 20 min. stabilization.
• Verify temp stability
Hot spot (5) = 247.36 oC
250 oC + 15 oC (USP
<1211>) over a 60 Cold spot (10) = 241.86 oC
minute period.
Dry Heat Depyrogenation – OQ
CFM = Average ft/min x area (ft2)
Air Velocity and Volume:
• Testing performed at ambient temperature.
• Airflow measured with thermal anemometer.
• Intake airflow measured at exhaust duct during
process and cool down phases.
• Circulation airflow measured at 5 equally spaced
locations on supply duct and exit duct walls approx.
15 cm. from wall and readings averaged.
• Performance to Manf.’s specifications.
Dry Heat Depyrogenation – OQ

Particle counts:
• Clean oven with IPA and lint-free wipes.
• Sample from open end of 10 mm SS tubing
placed at center of HEPA filter. Communicate
tubing to exterior of chamber via sanitary adapter
fitting and connect to particle counter. Measure
every 10 minutes at flow rate of 1 CFM.
• Performance consistent with Manufacturer's
specifications and intended application.
Dry Heat Depyrogenation – OQ

Chamber Pressure:
• Measure pressure of chamber during cycle via
sanitary port.
• Cycle temperature set at ambient to avoid injury.
• Differential pressure of > 0.03” W.C. relative to
atmospheric as per Manufacturer's specifications.
Dry Heat Depyrogenation – OQ

Confirm documentation:
• Verify successful IO/OQ
with signed report.
• Verify approved SOPs for
operation and cleaning.
• Verify maintenance and calibration schedule.
• Verify spare parts entered into facility’s system.
• Assemble all vendor related documentation.
Dry Heat Depyrogenation – HP
Cycle Development/Heat Penetration Studies:
• Prepare maximum load pattern.
• Calculate cycle parameters for desired process
conditions and application.
• Consider each item’s mass, complexity, cavities,
composition, density, etc.
• Place thermocouples in chamber and within load to
determine slow-to-heat zones within items being
processed. Thermocouples to be placed in slow-to-
heat zones during subsequent PQ testing.
Dry Heat Depyrogenation – Endotoxin Challenge
Endotoxin indicators:
• Prepared from CSE.
• > 1000 EU (50 – 200% var.).
• Direct product inoculation preferred but may (will)
present recovery issues.
• Usually prepared in 5 -10 sealed glass vials (ECV)
placed within load at cold spots.
• Positive and Negative controls.
Dry Heat Depyrogenation – Reduced Cycle

• Perform at least one cycle under reduced cycle

conditions (cycle setpoint reduced by 10 oC and
exposure time reduced by 10 minutes) using a
maximum load with thermocouples and endotoxin
challenge indicators to demonstrate the robustness
of the process (“worst case conditions”) and to
accommodate potential process variations and
variability in interpretation of process parameters.
Dry Heat Depyrogenation – PQ
• Place 12 TC’s in “worst
case position” within load
determined from HP
studies. Place one TC at
control probe, one TC at
center of chamber.
• Place endotoxin challenge
vials adjacent to TC’s in
load.
• Process 3 cycles.
Dry Heat Depyrogenation – PQ
• Calculate FH at 250 oC:

t (T – Tb)/Z

FH = 0 ∫ 10 dt
where:
t = time at end of hold period.
T = temperature (actual temperature).
Tb = baseline temperature (250 oC).
Z = z-value of endotoxin (46.41 – 54 oC2).
1Tsuji and Harrison. 1978. Appl. Environ. Mbio. 36(5): 710-713.
2Akers et. Al.1982. J. Parent. Sci. Technol. 36(1):23-27.
Dry Heat Depyrogenation – PQ
Calculate endotoxin log reduction value:

• LRVendotoxin = log (A) – log (B)

where: A = positive control
B = test sample

• LRVendotoxin = log (2200 EU/vial) – log (0.01 EU/vial)

= 3.34 EU – (– 2 EU)
= 5.34 EU
Dry Heat Depyrogenation – PQ
PQ Acceptance criteria:
• All TC’s within + 15 oC of setpoint (250 oC) during
hold period.
• Control probe within + 5 oC of adjacent TC at
midpoint of hold period.
• Cumulative FH (250 C) values > 30 minutes.
• All endotoxin challenge exhibit > 3 log reduction.
• Endotoxin positive controls > 1000 EU.
• Endotoxin negative controls indicate no residual
endotoxin contamination.
Dry Heat Depyrogenation – Done!
• All phases of validation successfully completed and
final report signed off.
• Review overall validation process and deviations to
determine how process could be handled better in
the future:
– Carefully write protocols and acceptance criteria, try to
anticipate problems or issues in advance.
– Coordination with other ongoing activities to ensure
required resources will be available when needed.
– Coordination with outside vendors.
– Ergonomics (maintenance/calibration).
Dry Heat Depyrogenation – Done!

• Allot extra time for

validation. It always
takes longer than you
think, particularly with
a new installation!