Sterilization validation
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The document provides an overview of sterilization validation processes for pharmaceutical products, including microbial growth kinetics and different sterilization methods such as moist heat, dry heat, ethylene oxide gas, and radiation. It details the stages of microbial growth, the importance of validation protocols, and specific technical aspects like D-values, Z-values, and F0 values relevant to ensuring effective sterilization. Additionally, it emphasizes the need for comprehensive validation of sterilization processes to maintain product safety and efficacy, along with routine monitoring and equipment calibration.
Sterilization validation
- 2. 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 (terminally sterilised) • those that cannot be terminally sterilised and must be aseptically prepared
- 3. Microbial Growth Kinetics •Microbial Growth Kinetics describe how the microbe grows in the fermenter. This information is important to determine optimal batch times. The growth of microbes in a fermenter can be broken down into four stages: • Lag Phase • Exponential Phase • Stationary Phase • Death Phase
- 4. Microbial Growth Kinetics •Lag Phase • This is the first phase in the fermentation process • The cells have just been injected into a new environment and they need time to adjust accordingly • Cell growth is minimal in this phase.
- 5. Microbial Growth Kinetics •Exponential Phase • The second phase in the fermentation process • The cells have adjusted to their environment and rapid growth takes place • Cell growth rate is highest in this phase
- 6. Microbial Growth Kinetics •Exponential Phase (Continued) • At some point the cell growth rate will level off and become constant • The most likely cause of this leveling off is substrate limited inhibition • Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying.
- 7. Microbial Growth Kinetics •Stationary phase • This is the third phase in the fermentation process • The cell growth rate has leveled off and become constant • The number of cells multiplying equals the number of cells dying
- 8. Microbial Growth Kinetics •Death phase • The fourth phase in the fermentation process • The number of cells dying is greater than the number of cells multiplying • The cause of the death phase is usually that the cells have consumed most of the nutrients in the medium and there is not enough left for sustainability
- 9. Sterilization - Overview What is the definition of “sterile”? • Free from microorganisms In practice no such absolute statement regarding absence of microorganisms can be proven • Defined as the probability of 1 in a million of a container being contaminated (10-6) • This referred to as the Sterility Assurance Level (SAL) • Organisms are killed in an exponential fashion
- 10. Sterilization - Overview Commonly used methods of sterilization • Moist Heat • Dry Heat • Gas (Ethylene oxide) • Radiation (Gamma or Electron) • Filtration • Others - UV, Steam and formaldehyde, hydrogen peroxide
- 11. Moist Heat • Saturated steam • Common cycles: • 121°C for 15 minutes • 134°C for 3 minutes • Other cycles of lower temperature and longer time may be used (e.g. 115°C for 30 minutes) • Used for sterilization of: • terminal sterilization of aqueous injections, ophthalmic preparations, irrigation & hemodialysis solutions, equipment used in aseptic processing • not suitable for non-aqueous/dry preparations • preferred method of sterilization
- 12. D- value: Time in mins., at a specific temp, to reduce surviving microbial population by 90% (1 logarithmic reduction) Z value : temp changed required, resulting in 1-log reduction (D-value) • F value : No. of mins. to kill a specified no. of MO with a specified Z- value at a specific temp • F 0 value : No of min to kill a specified no of MO with Z- value of 10 o C at a temp 121.1 o C • steam sterilization process must be sufficient to produce an F0 value of at least 8 min
- 13. Dry Heat • Lethality due to oxidative processes • Higher temperatures and longer exposure times required • Typical cycles: • 160°C for 120 minutes • 170°C for 60 minutes • 180°C for 30 minutes • tunnels used for the sterilisation of glass vials may use much higher temperatures (300°) for a much shorter period
- 14. Dry Heat • Used for: • glassware and product containers used in aseptic manufacture, non aqueous thermostable powders and liquids (oils) • also used for depyrogenation of glassware (250°C) • (Pyrogens - substances found in cell wall of some bacteria which can cause fever when introduced into the body)
- 15. Ethylene Oxide Gas • Either pure or in mixtures with other inert gases • Requires presence of moisture • Complex process • Typical cycles: • 1-24hours • 25-1200 mg/L gas • 25-65°C • 30-85% relative humidity • Used for: • heat labile product containers • surface sterilization of powders • Adequate aeration to reduce toxic residues
- 16. Radiation • Gamma rays generated by Cobalt 60 or Caesium 137 radionuclides; or • Accelerated electrons from an electron generator • 25 kilograys (kGy) usual dose • dose dependent on bioburden (resistance of organisms not predictable) • process must be properly validated • used for: • dry pharmaceutical products • heat labile product containers • can cause unacceptable changes
- 17. Filtration • Removes organisms from liquids and gasses • 0.2 - 0.22 micron for sterilization • composed of cellulose esters or other polymeric materials • filter material must be compatible with liquid being filtered • used for bulk liquids, gasses and vent filters
- 18. Validation - Overview • Selection of sterilzation process must be appropriate for product • terminal sterilization is the method of choice • moist heat (autoclaving) is the most common process used for terminal sterilization • product must not be affected by heat • container/closure integrity must be established • items being sterilised must contain water (if sealed) or material must allow for removal of air and penetration of steam for steam (moist heat) sterilization
- 19. Validation - Protocol • Validation protocol should include the following details for each sterilization process • process objectives in terms of product type, container/closure system, SAL required • specifications for time, temperature, pressure and loading pattern • description of all equipment and support systems in terms of type, model, capacity and operating range
- 20. Validation - Protocol Moist Heat continued: • performance characteristics of all equipment e.g. pressure gauges, valves, alarm systems, timers, steam flow rates/pressures, cooling water flow rates, cycle controller functions, door closure gasketing and air break systems and filters • methodology for monitoring performance of equipment and the process and labatory testing methodology • personnel responsible for all stages and final evaluation (should have experience and necessary training and be authorized)
- 21. Validation - Calibration • Laboratory testing should be performed by a competent laboratory, methodology should be documented • All instruments must be calibrated e.g. • temperature recorders and sensors • thermocouples • pressure sensors for jacket and chamber • timers • conductivity monitors for cooling water • flow metres for water/steam • water level indicators when cooling water is used • thermometers including those for thermocouple reference, chamber monitoring and laboratory testing
- 22. Validation - Calibration • Indicators should be calibrated • physical and chemical indicators should be tested to demonstrate acceptable response to time and temperature • biological indicators should be tested for count and time/temperature exposure response • for commercial indicators - test certificate with count and D-value and exposure response should be available. Results acceptable if verified “in house” periodically. • In house indicators must be fully characterized (D-value, identification) and appropriate for sterilization process All indicators should be appropriately stored and within expiry
- 23. 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 cool down phases Typical temperature profile of a heat sterilization process
- 24. Validation - Cycle Development Spore Strips - a narrow strip of fibrous paper impregnated with a bacterial spore suspension contained in a glassine envelope Spore Dots - Circular pieces of fibrous paper impregnated with the spore suspension Spore Suspensions - pure spore suspension of the desired challenge organism which can be inoculated onto the surface of a material Self contained units containing spore strips or suspensions and the media in which they are to be incubated (simple/convenient to use)
- 25. Validation of Sterilization Basic Principles • Installation Qualification (IQ) • Ensuring equipment is installed as per manufacturer’s specification • Operation Qualification (OQ) • Ensuring equipment, critical control equipment and instrumentation are capable of operating within required parameters • Performance Qualification (PQ) • Demonstrating that sterilizing conditions are achieved in all parts of sterilization load • Physical and microbiological
- 26. Validation - Equipment Installation Qualification • Ensuring equipment is installed as per manufacturer’s specification • considerations for new and existing equipment • specifications for the type of autoclave, construction materials, power supplies and support services, alarm and monitoring systems with tolerances and accuracy requirements • for existing equipment documented evidence that the equipment can meet process specifications
- 27. Validation - Equipment 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 • 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 run
- 28. Validation - Performance Performance Qualification • Demonstrating that sterilizing conditions are achieved in all parts of sterilization load • Physical and microbiological Physical • Heat distribution studies on empty chamber • maximum and minimum cycle times and temperatures • to identify heat distribution patterns including slowest heating points • mulitple temperature sensing devices should be used (thermocouples) • location of devices should be documented and ensure that heat distribution is uniform
- 29. Validation - Performance Performance Qualification - Physical (2) • Heat distribution of maximum and minimum chamber load configurations • multiple thermocouples throughout chamber (not inside product containers) to determine effect of load configuration on temperature distribution • temperature distribution for all loads using all container sizes used in production should be tested • position of thermocouples should be documented • Slowest to heat/cold spots in each run should be documented, inlcuding the drain • repeat runs should be performed to check variability • temperature distribution profile for each chamber load configuration should be documented
- 30. Validation - Performance Performance Qualification - Physical (3) • Heat penetration studies to detect the maximum and minimum temperature within all loads • all parts of each load must be on contact with steam • need to determine lowest and highest temperature locations 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 heat solution should be used • maximum and minimum load configurations for each sterilization cycle using routine cycle parameters
- 31. Validation - Performance Performance Qualification - Physical (4) • Heat penetration (2) • May be necessary for container mapping for larger volumes - cold spot then used for penetration studies • Need to consider effects of packaging e.g. overwrapping • Three runs performed once cold spots have been identified to demonstrate reproducibility
- 32. Validation - Performance Performance 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 Geobacillus stearothermophilus are most commonly used (considered “worst case”). BIs containing other organisms may be used • performance studies based on product bioburden require a considerable amount of work • indicators should be placed throughout the load, adjacent to thermocouples, at “cold spots” and slowest to heat locations (identified during heat penetration studies) • any growth is unacceptable unless processing errors demonsrated
- 33. Validation - Performance • Validation report must demonstrate requirements in Validation protocol have been met, any deviations must be justified • Requalification must be repeated on an annual basis (usually one run is acceptable) • Any changes or modifications must be evaluated • may just require requalification • any changes to loading patterns, new container/closure systems or cycle parameters require full validation
- 34. Routine Production Issues considered for routine production • Manufacturing environment should be controlled • Procedures in place to minimize the pre-sterilization bioburden • bioburden limits specified (although not so important when “overkill” cycle used) • Time between filling and sterilization should be specified • Integrity of container/closure system should be periodically verified • Periodic leak testing of chamber (if vacuum is part of cycle)
- 35. Routine Production • Cooling water should be sterile • Differentiation between sterilized and not-yet sterilized product • Physical separation (double ended autoclave) • Labelling and use of visual indicators (e.g autoclave tape) • Periodic testing of containers to verify integrity of container/closure system • Quality of steam should be defined and periodically tested for contaminants
- 36. Routine Production • Each sterilization cycle must be monitored • temperature, time and pressure recorded • temperature recorder independent from cycle controller • second independent temperature recorder • drain temperature should be recorded • chemical and biololgical indicators (if applicable) • Sterilisation cycle records should form part of batch records
- 37. Other Sterilization Processes Dry Heat • Should have air circulation in the chamber • Positive pressure in the chamber to prevent entry of non-sterile air • HEPA filtered air supplied • Biological indicators containing Bacillus atropheus (if used) • removal of endotoxin is usually sufficient • When removing pyrogens need to validate process using challenge tests
- 38. • B. Qualification and Calibration: • Mechanically Checking, Upgrading, and Qualifying the Sterilizer Unit : MAIN CONCERN : complete removal of air from the chamber and replacement with saturated steam .Modern autoclaves use cycles of vacuum and steam pulses to increase the efficiency of air removal. IQ/OQ must be done properly temperature and pressure instrumentation must be calibrated. • 2. Selection and Calibration of Thermocouples: • Copper constantan wires coated with Teflon are a popular choice. Accuracy of thermocouples should be ±0.5°C. 0.1°C in temperature measured by a faulty thermocouple will produce a 2.3% error in the calculated F0 value. Thermocouple accuracy is determined using National Bureau of Standards (NBS). • "Fo means the equivalent amount of time, in minutes at 121°C or 250 F, which has been delivered to a product by the sterilization process". For the practical calculation of F0, "a z-value of 10°C or 18 F is assumed; the term z- value. means the slope of the thermal death time curve and may be expressed as the number.
- 39. • 2. Selection and Calibration of Thermocouples cont...: • Thermocouples should be calibrated before and after a validation experiment at two temperatures: 0°C and 125°C. Any thermocouple that senses a temperature of more than 0.5°C away from the calibration temperature then it should be discarded. • 3. Selection and Calibration of BI (Biological Indicator): • B. Stearothermophilus spores are most commonly used BIs in validating steam sterilization cycles. main purpose of BIs is to assure that a minimum F 0 value has been achieved in the coolest location of the autoclave load D 121 and Z values of the BI must be accurately known. Spore strips or spore suspensions are used in the validation studies •
- 40. • 4. Container Mapping: • 4. Container Mapping A sufficient number of thermocouples should be positioned in areas representing the upper, middle, and lower portions of the container. Error in cold spot determinations may be introduced by employing an excessive number of thermocouples. Repeat studies are required to establish reproducible cold points and temperature profiles The profile point having the lowest temperature or lowest F0 is designated as the cold spot. The temperature profile of the container should remain constant among different sterilizing chambers, utilizing steam heat as the sterilizing medium. •
- 41. • C. Heat-Distribution Studies: • Heat-Distribution Studies 2 phases : in an empty autoclave chamber and in a loaded autoclave chamber 10 and 20 thermocouples are placed in definite arrangement • Teflon tape can be used to secure thermocouples wires should not make contact with the autoclave interior walls or any metal surface One thermocouple each should remain in an ice bath and high-temperature oil bath during each cycle The key is to identify the cool spot and the effect of the load size, configuration on the cool spot location The difference in temperature between the coolest spot and the mean chamber temperature should be not greater than ±2.5°C