Sterilization - validation,qualification requirementsDawn Tavalsky1ISPE – Boston ChapterFramingham MA 19Sep2013Sterilization - OverviewObjectives– Discuss definition of “Sterile”– Briefly describe sterilization methods– Describe approaches to be used for thevalidation of a sterilization process usingMoist Heat as an example– Describe requirements for routine monitoringand control of sterilization– Review issues that are specific to othersterilization processes21

Sterile Products - Overview Certain pharmaceutical products must be sterile– injections, ophthalmic preparations, irrigations solutions,haemodialysis solutions Two categories of sterile products– those that can be sterilised in final container (terminallysterilised)– those that cannot be terminally sterilised and must beaseptically prepared3Sterilization - OverviewWhat is the definition of “sterile”? Free from microorganismsIn practice no such absolute statement regarding absenceof microorganisms can be proven Defined as the probability of 1 in a million of acontainer being contaminated (10-6) This referred to as the Sterility Assurance Level(SAL) Organisms are killed in an exponential fashion42

Definition of “Sterile”MICROBIAL INACTIVATION-ktN t No e12003.503.0010002.50No of surviving organisms2.008001.501.006000.50No remaining0.00Log No remaining400-0.50-1.00200-1.500012345678910No remaining10003681355018721000Log No 91-1.34-2.00Time5Definition of “Sterile”Resistance of an organism is referred as its “D-value”D-ValueD-value - Time (or dose) required to reduce thepopulation of organisms by 1 log (or 90%)63

Definition of “Sterile” A sterilization process must deliver aSterility Assurance Level (SAL) of 1 in amillion (10-6) It is not possible to measure “10-6” The required SAL can be achieved byapplying a process that will reduce thenumber of organisms to zero and thenapply a safety factor that will deliver anextra 6 log reduction7Definition of “Sterile”Example For an initial bioburden of 102 thesterilization process will need toachieve an 8 log reduction inviable organismsThis will require 8 times the Dvalue (e.g. if the organism has aD value of 2 minutes then 8 x 2 16 minutes will be required toachieve an 8 log reduction andan SAL of 10-6) (Point Z) 84

Sterilization - OverviewCommonly used methods of sterilization––––––Moist HeatDry HeatGas (Ethylene oxide)Radiation (Gamma or Electron)FiltrationOthers - UV, Steam and formaldehyde,hydrogen peroxide9Moist Heat Saturated steam Common cycles:– 121 C for 15 minutes– 134 C for 3 minutes– Other cycles of lower temperature and longertime may be used (e.g. 115 C for 30 minutes) Used for sterilization of:– terminal sterilization of aqueous injections,ophthalmic preparations, irrigation &haemodialysis solutions, equipment used inaseptic processing105

Moist Heat not suitable for non-aqueous/drypreparations preferred method of sterilization11Dry Heat Lethality due to oxidative processes Higher temperatures and longer exposuretimes required Typical cycles:––––160 C for 120 minutes170 C for 60 minutes180 C for 30 minutestunnels used for the sterilisation of glass vialsmay use much higher temperatures (300 ) for amuch shorter period126

Dry Heat Used for:– glassware and product containers used inaseptic manufacture, non aqueousthermostable powders and liquids (oils) also used for depyrogenation of glassware( 250 C)– (Pyrogens - substances found in cell wall ofsome bacteria which can cause fever whenintroduced into the body)13Ethylene Oxide Gas Either pure or in mixtures with other inertgases Requires presence of moisture Complex process Typical cycles:––––1-24hours25-1200 mg/L gas25-65 C30-85% relative humidity147

Ethylene Oxide Used for:– heat labile product containers– surface sterilization of powders Adequate aeration to reduce toxicresidues15Radiation Gamma rays generated by Cobalt 60 or Caesium137 radionuclides; or Accelerated electrons from an electron generator 25 kilograys (kGy) usual dose– dose dependent on bioburden (resistance of organismsnot predictable) process must be properly validated used for:– dry pharmaceutical products– heat labile product containers can cause unacceptable changes168

Filtration Removes organisms from liquids andgasses 0.2 - 0.22 micron for sterilization composed of cellulose esters or otherpolymeric materials filter material must be compatible withliquid being filtered used for bulk liquids, gasses and ventfilters17Validation - Overview Selection of sterilzation process must beappropriate for product– terminal sterilization is the method of choice– moist heat (autoclaving) is the most commonprocess used for terminal sterilization– product must not be affected by heat– container/closure integrity must be established– items being sterilised must contain water (ifsealed) or material must allow for removal ofair and penetration of steam for steam (moistheat) sterilization189

Validation - ProtocolRequirements for Moist Heat SterilizationOther processes follow similar requirements Validation protocol should include thefollowing details for each sterilizationprocess– process objectives in terms of product type,container/closure system, SAL required– specifications for time, temperature, pressureand loading pattern– description of all equipment and supportsystems in terms of type, model, capacity andoperating range19Validation - ProtocolMoist Heat continued:– performance characteristics of all equipmente.g. pressure gauges, valves, alarm systems,timers, steam flow rates/pressures, coolingwater flow rates, cycle controller functions,door closure gasketing and air break systemsand filters– methodology for monitoring performance ofequipment and the process and labatorytesting methodology– personnel responsible for all stages and finalevaluation (should have experience andnecessary training and be authorized)2010

Validation - Calibration Laboratory testing should be performedby a competent laboratory, methodologyshould be documented All instruments must be calibrated e.g.––––––––temperature recorders and sensorsthermocouplespressure sensors for jacket and chambertimersconductivity monitors for cooling waterflow metres for water/steamwater level indicators when cooling water is usedthermometers including those for thermocouplereference, chamber monitoring and laboratory testing21Validation - Calibration Indicators should be calibrated– physical and chemcial indicators should be testedto demonstrate acceptable response to time andtemperature– biological indicators should be tested for countand time/temperature exposure response for commercial indicators - test certificate with countand D-value and exposure response should beavailable. Results acceptable if verified “in house”periodically. In house indicators must be fully characterized (Dvalue, identification) and appropriate for sterilizationprocessAll indicators should be appropriately stored andwithin expiry2211

Validation - Cycle Development Concept of Fo– Lethality factor equivalent to time at 121 C 1 minute at 121 C is equivalent to Fo of 1.Lethality can accumulate during heat up and cooldown phasesTypical temperature profile of a heat sterilization processWhat would be the Fo of a cycle at 121 C for 15 minutes?23Validation - Cycle Development Fo is calculated using the followingequation:Fo tΣ10(T-121/Z)where: “ t” is the time interval between measurements oftemperature (T) “T” is the temperature of sterilised product at time (t) “Z” is a temperature coefficient which measures thenumber of degrees required to change the D-value ofan organism by 1 log2412

Validation - Cycle Development The minimum Fo required by a sterilzationprocess is related to the resistance of thebioburden (D-value)Fo D121 (LogA - Log B)where: “D121” is equal to the time at 121 C to reduce thepopulation of the most resistant organism in eachproduct container by 90% (or 1 log) “A” is the number of microoganisms per container “B” is the maximum acceptable probability ofsurvival (Sterility Assurance Level , 10-6)25Validation - Cycle Development Two approaches to sterilization– Overkill– Probability of survival Overkill approach used when the productcan withstand excessive heat treatmentwithout adverse effects– Cycle should deliver an Fo of at least 12 This will achieve a 12 log reduction ofmicroorganisms with a D-value of 1 minute(Assuming each product unit contains 106 organisms a 12log reduction will result in 10-6 organisms per unit orprobability of survival (SAL) of 1 in a million)(Normal bioburden is usually much lower and the organismsnormally much less resistant than this)2613

Validation - Cycle DevelopmentBiological Indicators device consisting of a known number ofmicroorganisms, of a known resistance to aparticular sterilization process in or on a carrierand enclosed in a protective package.– Organisms are in the form of endospores (not vegetativestate) as these are most resistant to sterilization27Validation - Cycle DevelopmentSpore Strips - a narrow strip offibrous paper impregnated with abacterial spore suspensioncontained in a glassine envelopeSpore Dots - Circular pieces offibrous paper impregnated withthe spore suspensionSpore Suspensions - pure sporesuspension of the desiredchallenge organism which can beinocluated onto the surface of amaterialSelf contained units containing sporestrips or suspensions and themedia in which they are to beincubated (simple/convenient touse)2814

Validation - Cycle Development Probability of Survival approach used usedfor heat labile productsThe process is validated toachieve a destruction of thepresterilization bioburden toa level of 100 (Point Y), with aminimum safety factor of anadditional six-log reduction(Point Z)Determination of theminimum Fo required isbased on the bioburden andits heat resistanceWhat Fo would be required for a bioburden of 102 (100) if D-value was 1?29Validation of SterilizationBasic Principles Installation Qualification (IQ)– Ensuring equipment is installed as permanufacturer’s specification Operation Qualification (OQ)– Ensuring equipment, critical control equipmentand instrumentation are capable of operatingwithin required parameters Performance Qualifcation (PQ)– Demonstrating that sterilizing conditions areachieved in all parts of sterilization load– Physical and microbiological3015

Validation - EquipmentInstallation Qualification Ensuring equipment is installed as permanufacturer’s specification– considerations for new and existing equipment– specifications for the type of autoclave,construction materials, power supplies andsupport services, alarm and monitoringsystems with tolerances and accuracyrequirements– for existing equipment documented evidencethat the equipment can meet processspecifications31Validation - EquipmentOperational Qualification Ensuring equipment, critical controlequipment and instrumentation are capableof operating within required parameters Three or more test runs which demonstrate– controls, alarms, monitoring devices and operationindicators function– chamber pressure integrity is maintained– chamber vacuum is maintained (if applicable)– written procedures accurately reflect equipment operation– pre-set operation parameters are attained for each run3216

Validation - PerformancePerformance Qualification Demonstrating that sterilizing conditions areachieved in all parts of sterilization load Physical and microbiologicalPhysical Heat distribution studies on empty chamber33– maximum and minimum cycle times and temperatures– to identify heat distribution patterns including slowestheating points– mulitple temperature sensing devices should be used(thermocouples)– location of devices should be documented and ensurethat heat distribution is uniformValidation - PerformancePerformance Qualification - Physical (2) Heat distribution of maximum and minimumchamber load configurations– multiple thermocouples throughout chamber (not insideproduct containers) to determine effect of loadconfiguration on temperature distribution– temperature distribution for all loads using all containersizes used in production should be tested– position of thermocouples should be documented– Slowest to heat/cold spots in each run should bedocumented, inlcuding the drain– repeat runs should be performed to check variability– temperature distribution profile for each chamber loadconfiguration should be documented3417

Validation - PerformancePerformance Qualification - Physical (3) Heat penetration studies to detect the maximumand minimum temperature within all loads– all parts of each load must be on contact with steam– need to determine lowest and highest temperaturelocations and slowest and fastest to heat locations(measured inside product containers)– need to consider all variables such as container size,design, material, viscosity of solution and fill volume.Container with maximum fill volume and slowest to heatsolution should be used– maximum and minimum load configurations for eachsterilization cycle using routine cycle parameters35Validation - PerformancePerformance Qualification - Physical (4) Heat penetration (2)– May be necessary for container mapping for largervolumes - cold spot then used for penetration studies– Need to consider effects of packaging e.g. overwrapping– Three runs performed once cold spots have beenidentified to demonstrate reproducibility3618

Validation - PerformancePerformance Qualification - Microbiological Biological challenge studies– used when Probability of Survival approach is used– may not be necessary when cycle is 121 C for 15 minutes(except US and Australia)– biological indicators (BI) containing spores of Geobacillusstearothermophilus are most commonly used (considered“worst case”). BIs containing other organisms may be used– performance studies based on product bioburden require aconsiderable amount of work– indicators should be placed throughout the load, adjacent tothermocouples, at “cold spots” and slowest to heat locations(identified during heat penetration studies)– any growth is unacceptable unless processing errorsdemonsrated37Validation - Performance Validation report must demonstrate requirementsin Validation protocol have been met, anydeviations must be justified Requalification must be repeated on an annualbasis (usually one run is acceptable) Any changes or modifications must be evaluated– may just require requalification– any changes to loading patterns, new container/closuresystems or cycle parameters require full validation3819

Routine ProductionIssues considered for routine production Manufacturing environment should be controlled Procedures in place to minimize the presterilization bioburden– bioburden limits specified (although not so importantwhen “overkill” cycle used) Time between filling and sterilization should bespecified Integrity of container/closure system should beperiodically verified Periodic leak testing of chamber (if vacuum is partof cycle)39Routine Production Cooling water should be sterile Differentiation between sterilized and not-yetsterilized product– Physical separation (double ended autoclave)– Labelling and use of visual indicators (e.gautoclave tape) Periodic testing of containers to verify integrity ofcontainer/closure system Quality of steam should be defined andperiodically tested for contaminants4020

Routine Production Each sterilization cycle must be monitored– temperature, time and pressure recorded– temperature recorder independent from cyclecontroller– second independent temperature recorder– drain temperature should be recorded– chemical and biololgical indicators (ifapplicable) Sterilisation cycle records should formpart of batch records41Other Sterilization ProcessesSterilization using other processes shouldfollow a similar approach as thatdescribed for moist heat– Validation protocol– Equipment calibration– Determining the process that will deliver thedesired SAL (10-6)– IQ, OQ, PQ– Requirements for routine monitoring andcontrol4221

Other Sterilization ProcessesDry Heat– Should have air circulation in the chamber– Positive pressure in the chamber to prevententry of non-sterile air– HEPA filtered air supplied– Biological indicators containing Bacillusatropheus (if used) removal of endotoxin is usually sufficient– When removing pyrogens need to validateprocess using challenge tests43Other Sterilization ProcessesRadiation– Usually performed by contracting service(need to ensure validation status,responsibilities)– Based on bioburden of product beingsterilised Biological indicators may be used as additionalcontrol but may not be as resistant as naturallyoccuring bioburden– Method defined in International Standard ISO111374422

Other Sterilization ProcessesRadiaton (2)– Correct dose of radiation ( 25 kGy) receivedby all products (measured with dosimeters) quantitative measurement number, location, within calibration time-limit– Radiation sensitive colour discs applied topackaging– procedures to distinguish irradiated and nonirradiated materials– Variation in density of packaging should beaddressed during validation45Other Sterilization ProcessesGasses and Fumigants– e.g. ethylene oxide, hydrogen peroxide vapour– Only when no other method is suitable– Must demonstrate that process does notadversely affect product– Packaging must be able to permit ingress ofgas and humidity– Ensure product load is adequately heated andhumidified prior to sterilization (called“conditionning”) need to take into account validation performed insummer or winter4623

Other Sterilization ProcessesGasses and Fumigants (2)– Temperature distribution is acceptable– Concentration of sterilant gas is sufficient– Use of biological indicators is important(Bacillus atropheus)– Half cycles If cycle of half normal time destroys biological indicators(106 organisms), double time will achieve SAL of 10-6– Aeration Ventilated conditions Defined limits of residuals Process included in validation– Safety and toxicity issues considered47Useful Publications ISO/EN 17665 - Sterilization of health careproducts - Moist Heat (Parts 1 and 2) ISO/EN 11135 - Sterilization of health careproducts - Ethylene Oxide (Parts 1 and 2) ISO/EN 11137 - Sterilization of health careproducts - Radiation (Parts 1,2 and 3) “Validation of Moist Heat Sterilization Processes:Cycle Design, Development, Qualification andongoing Control. PDA Technical Report No. 1Revised 20074824


Sep 19, 2013 · Operational Qualification Ensuring equipment, critical control equipment and instrumentation are capable of operating within required parameters Three or more test runs which demonstrate – controls, alarms, monitoring devices and operation indicators function –