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Photo courtesy of Texwipe

DRY HEAT DEPYROGENATION CONSIDERATIONS:

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

Patrick J. McCormick, [email protected]+ 1 585 338 8390

mailto:[email protected]

Pyrogens and Endotoxin

• Sterilization and depyrogenation seldom drive the development of new products, but are essential to the safety of our products and the health of our customers.

Endotoxin tied to sterilizers. Hospital traces fatal outbreak. Saturday, December 16, 1995.Cookson, S.T. et al. Pyrogenic Reactions in Patients Undergoing Cardiac Catherization Associated with Contaminated Glass Medicine Cups. Catherization and Cardiovascular Diagnosis 42:12-28(1997)

Outbreak traced to enzyme detergent contaminated with > 104 CFU/ml; 434 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 EndotoxinEndotoxin:• 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 order inactivation kinetics.

• z = 20 oC.• FH at 170 oC.• Total kill of 106

spores of Bacillus atrophaeus at half cycle based on overkill approach of ISO 14937/14161. 0.000001

0.000010.0001

0.0010.01

0.11

10100

100010000

1000001000000

exposure

10-6

SAL

1/2 cyclewindow

63%pos.

1% pos.

Dry Heat Depyrogenation - Kinetics

250 oC180 oC30 minutes

None170 oC60 minutes

None160 oC120 minutes

DepyrogenationSterilizationDry heat exposure*

* Actual exposure time will vary with load and conditions.

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 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).


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


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?


• 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?


• 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?


• 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).


• 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.


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 StudyOven 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.


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


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?


• Positioning of unit.• Leveling/anchoring.• Baffles and panels.• Seals and gaskets.• Insulation.• Utilities (air, water,

electrical, etc.).

• Ducting and plumbing.• HEPA filter installation

and certification.• Environmental and

Safety inspection.• Manuals and drawings.• Spare parts list.

Confirm physical installation to specifications:


Calibration equipment:• Tachometer.• Temperature calibrator.• Thermocouple bath.• Data logger.• Pressure calibrator.• Thermal anemometer.• Particle counter.

Oven Instrumentation:• Temperature control.• Chart recorder.• High limit alarm.• Intake pressure.• Exhaust pressure.• Recirculation pressure.• Chamber pressure.

Verify Calibration:


• 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…


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.


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.


Empty chamber test:• Place 12 TC’s as

indicated w/in 8 cm of chamber surface.

• Oven set at 250 oC.• 20 min. stabilization.• Verify temp stability

250 oC + 15 oC (USP <1211>) over a 60 minute period.

Cold spot (10) = 241.86 oC

Hot spot (5) = 247.36 oC

Avg. = 244.61 oC


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.

CFM = Average ft/min x area (ft2)


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.


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.


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 – PQPQ 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!

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Documents

endotoxin endotoxin

oc depyrogenation

dry heat exposure

endotoxin sterilization

depyrogenation ovens

reference endotoxin

endotoxin pyrogen

accepted endotoxin limit