VALIDATION OF MOIST HEAT STEILIZATION

1. VALIDATION OF MOIST HEAT STEILIZATION

2. Definition Sterilization “The act or process, physical or chemical, that destroys or eliminates all viable microbes including resistant bacterial spores from a fluid or a solid.” Examples of sterilization methods are : steam treatment at 121℃, dry heat at 230℃, flushing with a sterilizing solution such as hydrogen peroxide (H2O2) or ozone (O3), irradiation, and filtration. Sterility “The reduction of anticipated levels of contamination in a load to the point where the probability of survival is less than 10-6.”

3. Definition D-value The time in minutes required for a one-log or 90% reduction of a specific microbial population under specified lethal conditions. For steam sterilization it is determined at a constant temperature z-value The number of degree of temperature change necessary to change the D-value by a factor of 10.

4. Definition F value(lethal rate, instantaneous Fo) The F value is a measurement of sterilization effectiveness. F(T,z) is defined as the equivalent time at temperature T delivered to a container or unit of product for the purpose of sterilization, calculated using a specific value of z. Fo value(accumulated Fo) The term "Fo " is defined as the number of equivalent minutes of steam sterilization at temperature 121.1°C delivered to a container or unit of product calculated using a z-value of 10°C. Fo =  10^((121-T)/z)*t

5. Methodology Overkill Sterilization Provides a minimum 12 log reduction of a resistant BI w/ a known D-value of not less than 1 minute. Required minimal information on the bioburden Bioburden/Bioindicator Sterilization Provides a probability of survival of less than 1 in 106 for the bioburden as demonstrated using a resistant BI w/ a known D-value. BI may not be inactivated Requires information on the numbers and heat resistance of the BI. Requires ongoing monitoring or control over bioburden.

6. Methodology Bioburden Sterilization Provides a probability of survival of less than 1 in 106 for the most resistance bioburden expected in the load. Requires information on the numbers and heat resistance of the BI. Requires ongoing monitoring or control over bioburden.

7. Sterilizer Cycle Gravity Displacement Difference of density Density of air at 20℃ = 1.2 g/ℓ Density of steam at 100℃ = 0.6 g/ℓ Effectiveness of air elimination depends on the rate of steam supply Air pocket : too rapidly Diffusion into the steam : too slowly, more difficult to remove Specially designed steam trap permitting the passage of large volume of air

8. Sterilizer Cycle Prevacuum cycle A more effective method By means of a mechanical vacuum pump or a steam eductor Vacuum as low as 15~20mmHg, apply for 8~10 min. Pulsing cycle A series of alternating steam pulses followed by vacuum excursions Air-steam mixture Terminal sterilization of large volume parenterals Air injection required to compensate the great expansion of air or nitrogen in the head space above the liquid Well mixed chamber : fan, raining effect by external pump w/ cooling

9. Cycle Development Consider factors into account Nature of the load : porous materials, heat sensitivity of the products Type of the sterilizer Employed containers and closures Heat stable product : overkill approach Heat liable product : bioburden approach Bioburden studies : number of microorganisms D-value studies : only highly resistant spore formers, BIER(biological indicator evaluator resistometer) Inoculate the spore into the actual solutions For solid materials, precut strips

10. Preparing for Validation Temperature sensing devices :  T type thermocouples(copper-constantan) encased in flexible sheaths  Premium grades of wire having 0.1℃ accuracy Temperature standards  RTD traceable to the National Bureau of Standards , IPR, HTR Calibration of thermocouples At two temperatures : 0 ℃, 130 ℃ Correction factors Stability : 0.03℃ Accuracy : 0.5℃

11. Preparing for Validation Autoclave Validation nozzle and adaptor Data logger : digital output and multi-channel device BIs or biological challenges Loads

12. Validation Protocol Protocol should include Objectives of the validation Responsibilities of validation personnel and operating department personnel Identification and description of the sterilizer and its process control Identification of SOPs :equipment Calibration of instrument : SOPs and/or description Identification and calibration of the temperature monitoring equipment

13. Validation Protocol A description of the following studies Bioburden determination studies(if applicable) Empty chamber heat distribution studies Container mapping studies(if applicable) Loaded chamber heat penetration studies Microbiological challenge studies Evaluation of drug product cooling water(if applicable) Integrity testing of vent filter Acceptance criteria References Review and approval

14. Heat Distribution Studies To demonstrate the temperature uniformity and stability of the sterilizing medium throughout the sterilizer Conduct on both the empty and loaded chamber with max. and min. load configurations Acceptance criteria : Less than ±1℃of the mean temperature Conduct 3 runs to obtain consistent results Distribution of the thermocouples : geometrical representatives, exhaust drain, adjacent to the control sensor At least 10 probes, normally 15~20 probes

15. Heat Distribution Studies At loaded chamber heat distribution test, the thermocouples should be positioned in the same locations used for empty chamber heat distribution Avoid contacting solid surfaces Do not place within any containers Data should be obtained at regular intervals

16. Container Mapping To determine the coolest point within the liquid filled container Temperature mapping should be conducted on all the different container types, sizes and fill volume to be validated The number of the thermocouples used depends on the container volume Possible to use a single thermocouple at different positions, and can be conducted in a smaller autoclave or retort Penetration thermocouples should be positioned at the cold spot having lowest temperature or Fo

17. Heat Penetration Studies To determine the coolest point(s) within the specified load and configuration, and to assure that these points be consistently exposed to sufficient heat lethality Prior to conduct heat penetration studies, determine max. and min. load configurations Probed container at the cold spot should be distributed uniformly throughout the load Penetration thermocouple are positioned at points within the process equipment suspected to be the most difficult for steam heat penetration

18. Heat Penetration Studies Lethal rate can be determined from the temperature data by the following formula : L = log-1(To-Tb)/z = 10^((To-Tb)/z) A summation over time of the lethal rate at a series of temperature(accumulated lethality) Fo =  10^((121-T)/z)*t Regard to product stability

19. Microbial Challenge Studies Biological challenges are employed during heat penetration studies in order to demonstrate the degree of process lethality provided by the sterilization cycle Microorganism frequently utilized Overkill : Bacillus stearothermophilus and Clostridium sporogenes Bioburden : Calibrated BIs from environmental and process isolates such as E. coli Type of BI : Spore strips or spore suspension into the suspending medium Microbial challenge studies are conducted concurrently with the heat penetration studies

20. Validation Report Common elements of all reports : Identification of the task report by number Reference to protocol A brief summary of the range of operational conditions experienced and how they were controlled A procedure for maintaining control within the approved range A summary and analysis of the experimental results A brief description of any deviation Conclusion Review and approval Cycle development reports are not usually a part of the validation report

21. Maintenance of Validation A routine calibration program for all instruments critical to the operation of the sterilizer and its support system A preventative maintenance program including periodic operational rechecks and comparison to OQ record Routine monitoring of bioburden and periodic BI challenges(optionally) Operating records and equipment logs Process and equipment change control procedures including review to establish whether additional validations are required On-going validation

22. Controversial Issues Incubation of the sterility test : 7 days vs. 14 days USP provide information concerning critical parameters for Parameteric Release Reduction extent and frequency of revalidation Verification of D-value of BIs Use of alternative to B. stearothermophilus as a BI