validation of Sterilization process
- 2. LARAIB JAMIL M.Phill Registered scholar Department of Pharmaceutics University Of Balochistan
- 3. Validation • Validation may be defined as : Establishing documented evidence which provides high degree of assurance that specific process will be consistently produce a product meeting its predetermined specifications & quality attributes.
- 4. FDA published guidelines • 3 principal should involve in validation process: 1. To build sterility into a product. 2. To demonstrate maximum level of probability that sterilization methods has established sterility to all batches of unit. 3. To provide greater assurance of result of end product sterility test.
- 5. 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.” • Sterility: “The reduction of anticipated levels of contamination in a load to the point where the probability of survival is less than 10⁻⁶.” • This referred to as STERILITY ASSURANCE LEVEL (SAL)
- 6. Commonly used sterilization methods are • Dry heat • Moist heat • Gas (Ethylene oxide, Hydrogen peroxide) • Radiation (Gamma or electron) • Others (UV, Steam & Formaldehyde, Filteration )
- 7. Process of microbial destruction 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 Importance of D-value : 1. It is specific kinetic expression of each microorganism. D-value will be effected by • Type of microorganism used as biological indicator • The formulation component & characteristics (e.g- Ph) • The surface on which microorganism is exposed (e.g- glass, plastic, in sol, in dry powder etc)
- 8. 2. Knowledge of D-value Is necessary for calculation of Z-value 3. The D-value is also used in calculations of F-value
- 9. • Z-value: is a term used in microbial thermal death time calculations. It is the number of degrees the temperature has to be increased to achieve a ten fold (i.e. 1 log10) reduction in the D-value. • F-value: The F value is a measurement of sterilization effectiveness. It is defined as number of minutes to kill number of microorganism with specified Z-value at a specific temperature
- 10. • Fₒ-value: It is saturated steam sterilization process in which the equivalent amount of time, in minutes at 121°C or 250 F, which has been delivered to a product’s final container for sterilization process with reference to microorganism possessing a Z-value of 10 (BP-2009) In general to aqueous preparation in steam deliver cycle the Fₒ value for not less than 8 to every container in autoclave is considered satisfactory (IP- 2007)
- 12. Validation of steam sterilization Describes sterilization techniques that utilize hot air that is heavily loaded to facilitate efficient sterilization by steam and pressure. Its validation studies include: A. Qualification & calibration 1) Mechanically checking, upgrading, qualifying the sterilizing units 2) Selection & calibration of thermocouples 3) Selection & calibration of BI 4) Container mapping B. Heat distribution studies C. heat penetration studies
- 13. A-Qualification & calibration Complete removal of air from chamber & replacement with saturated steam Older autoclave relied on gravity displacement Modern autoclave use cycles of vacuum & steam pulses to increase the efficiency of air removal Temperature & pressure instruments must be calibrated 1- Mechanically checking , upgrading & qualifying the sterilizer unit
- 14. 2- Selection & calibration of thermocouple • Copper constant wires coated with taflons are popular choice • Accuracy of thermocouples should be ±0.5⁰C (0.1⁰C in temp by faulty thermocouple will produce 2.3% error in calculation of fₒ value) • Thermocouple accuracy is determined using National Bureau of Standards (NBS) traceable constant temperature calibration instruments.
- 15. • Thermocouple should be calibrated before & after validation experiment at two temperature (0ᵒC & 125ᵒC) • Temperature sensed more then 0.5ᵒC by thermocouples should be discarded.
- 16. • B-Stearothermophillus spores are most commonly used BIs in steam sterilization process • Spores strips & spore suspensions are used in validation studies. 3- Selection & calibration of BI
- 17. • A sufficient number of thermocouples should be positioned in areas representing upper, lower, middle portions of container • Error in cold spot determination may be introduced by employing excessive number of thermocouples • Repeat studies are required to establish cold point & temp profiles • The temp profile of container should remain constant among different sterilizing chamber in which sterilizing medium is steam heat 4- Container mapping
- 18. B- Heat distribution studies • 2 PHASES : 1) In empty autoclave chamber 2) In loaded autoclave chamber 10-20 thermocouples are placed in definite position
- 19. • Teflon tape can be used to secure thermocouples • Wire should not make contact with autoclave walls/ any metal surface • Thermocouple should remain in ice-bath & high temperature oil-bath during each cycle • The key is to identify cool spot, the effect of load size, configuration on cool spot location • Note: the mean temp difference b/w cool spot & chamber difference should not be greater than ±2.5⁰C
- 20. C- Heat penetration studies • It is most critical component in which the container’s cold spot for containers ≥100ml is determined using container’s mapping studies • Thermocouples are inserted in the container & outside the container to establish cold point at constant temp baths • Minimum & maximum loading configuration should be studied • The effect of load-load variation on time, temp profile must also be determine
- 21. • GOALS: The goals are to ensure that validation cycle will remain unchanged in future. Any change in load size, load configuration or container characteristics must accompanied by repeat validation studies to prove that cool spot location has not changed
- 22. VALIDATION OF DRY HEAT STERILIZATION
- 23. Validation of dry heat sterilization • Dry heat, as the name indicates, utilizes hot air that is either free from water vapor, and where this moisture plays minimal or no role in the process of sterilization. • it includes Batch oven validation tunnel sterilizer validation a) Air balance distribution b) Heat distribution studies c) Heat penetration studies d) Mechanical reliability Biological process validation of dry heat sterilization Endotoxin challenge in the validation of dry heat sterilization
- 25. Batch Oven Validation • Air balance determination • Heat distribution of an empty chamber • Heat penetration studies • Mechanical reliability Mechanical reliability in terms of air velocity, temp consistency, reliability & sensitivity of all the oven & instrumental control must be verified.
- 26. Tunnel sterilizer validation • Extra variable is belt speed • This variable should be held constant by maintaining the same belt speed throughout validation process . Other variables are: 1. Air balance determination 2. Heat distribution of an empty chamber 3. Heat penetration studies 4. Mechanical reliability
- 27. Tunnel sterilizer validation Air Balance Determination: • Proper air balance is more critical than batch oven, because items being sterilized are in moving state & are exposed to different air system (ex- heating zone, cooling zone) • Major problem in tunnel sterilizer is control of particle because belt & items are natural source of particulate so air must be particulate free. Heat distribution studies: • It should determine where the cold spot is located as a function of width of belt & height of tunnel • Thermocouple must be long enough to be transported through the entire tunnel • Peak temperature should remain within ±10ᵒC across the belt for at least 3 replicate runs
- 28. Tunnel sterilizer validation Heat penetration studies: • Thermocouples should be placed at or nearest coolest point in container • The coolest point in container is at the junction of bottom of container & side walls • The container’s inner wall should be in contact with thermocouple’s tip because the objective is to sterilize the inner walls of container as well as inner space • 3-5 replicate runs for each commodity size & every loading should be done Mechanical reliability: • Air velocity, air particulate, temp consistency, reliability of all tunnel controls (heat zone temp, belt speed, blower functions) • Must be proved mechanical repeatability during physical validation studies
- 29. Biological process validation of dry heat sterilization Microorganisms are more resistant to dry heat so it is necessary to prove the dry heat ability to destroy MO & microbial endotoxin. Validation of dry heat sterilization should be based on destruction of endotoxin rather than on MO because of dry heat resistance of endotoxins.
- 30. Endotoxin validation of dry heat sterilization • Inoculate sample with known amount of endotoxin e.g- 10-1000mg E.coli pollysachrides • Thermocouples should placed in commodities adjacent to those containing endotoxin for temp monitoring & correlation with LAL test results. • Endotoxin destruction should be ascertained(to investigate) at the coolest location of the load. • Several endotoxin challenge should be done per cycle & study must be repeated 3-5 times
- 32. Gaseous sterilization The STERILIZATION BY THE APPLICATION OF GAS TYPES: • Validation of ethylene oxide sterilization cycle • Validation of vapor phase hydrogen peroxide sterilization process. 5 variables: - Heat - Ethylene oxide concentration - Relative humidity - Temperature - pressure
- 33. Procedure of ethylene oxide validation ADRESS THE PRODUCT SPECIFICATION & PACKAGING DESIGN • Chemical nature of component of product • Exit point: long/narrow lumens that will represent barriers to EtO permeation • How dense the material is? • What is the nature of primary or secondary packaging ? • Where are dead air spaces within package & within the load ? • Use laboratory sized EtO sterilizer during early stages of validation process. • Verify the calibration of all instruments involved in monitoring of EtO cycle.
- 34. Ethylene oxide sterilization PROCEDURE: • Perform heat distribution studies by using empty sterilizer • Identify the zones of temperature extremes • Most common biological indicator for EtO cycle = B.Subtilis Var. negar • Conc of these spores per strip should 10⁶ • By running empty sterilizer determine the accuracy & reliability of sterilizer
- 35. Ethylene oxide sterilization • Now start heat distribution & heat penetration studies using a loaded EtO sterilizer. The data should verify the following question 1. What is conc of EtO released in vessel? 2. What is conc of water vapor in vessel? 3. What is range of temp distribution throughout loaded vessel? 4. How much EtO is consumed during the cycle? 5. What are rates of creating vacuum/pressure? 6. Does the selected cycle sterilize the product Test should be conducted on final packaging Documentation should be integrated into single protocol.
- 36. Validation of vapor phase hydrogen peroxide sterilization process VPHP is relatively new gaseous sterilization process. It has advantages over other agents that : i. It doesn’t require temp above ambient. ii. There are no concern about residual by- products. The basic step in process are dehumidification, conditioning, sterilization & aeration.
- 37. Hydrogen peroxide sterilization process 5 steps that are part of validation studies: • Cycle development • Temperature distribution • Vapor distribution • Biological challenge • Aeration verification
- 38. Hydrogen peroxide sterilization process Cycle development parameters include • Temp • Airflow rate • Humidity • Hydrogen peroxide concentration • hydrogen peroxide delivery rate Temperature studies include use of temp. Vapor distribution studies uses chemical indicators. Biological challenge involves placement of biological indicators normally Bacillus stearothermophilus spore strips. Aeration verification determines the parameters (e.g- time, air exchange rate)
- 40. Validation of radiation sterilization process Is defined as dose of radiation necessary to produce a 90% reduction in a number of indicator microbial cells. It depends on factors such as temperature Moisture Organism species Chemical environment/physical surface on which indicator microorganism is attached. Bacillus Pumilus spores are USP choice as biological indicator for radiation sterilization.
- 41. 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 NOTE : Cycle development reports are not usually a part of the validation report