Autoclaves and Autoclave Validation

2. Autoclaves and Autoclave Validation TJ Stough June 8, 2010 Confidential

3. Presentation Overview • References • Autoclaves Defined • Types of Autoclaves • Cycle Types • Validation • Lethality and Sterility Assurance Level • Revalidation • Safety Confidential

4. PDA Technical Report #1, August 2007 Guidance for Industry For the Submission of Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products (CDER 1994) The Validation and Periodic Testing of Benchtop Vacuum Steam Sterilizers (MHRA DB 9804) References Confidential

5. Autoclaves Defined • Autoclaves are sterilizers which, using the physical properties of steam as a sterilant, combine the use of pressure and vacuum to ensure that the maximum benefit is gained from the sterilant in the shortest amount of time • Steam is superheated to provide the most efficient heat transfer

6. Why Autoclaves? • Autoclaves provide the most cost-effective and efficient form of sterilization for products and production components that are not heat or moisture sensitive. • Autoclaves leave no chemical residue compared to other forms of sterilization Confidential

7. Moist or Dry? It matters. • Steam – or moist heat in an autoclave kills by: • Coagulating and denaturing enzymes and structural protein • Penetrates cell walls • Does it in 15-30 minutes at 121°C • Dry heat – Convection or oven heat kills by: • destructive oxidation of essential cell constituents • Does it in 300 minutes at 121°C or ~ 60 minutes at 160°C • Cannot be used for liquids • Usually only used for glass and metal. Confidential

8. Types of Autoclaves Benchtop Autoclaves (gravity) • Found in dental offices, tattoo shops, etc. • Advantages • Easy to operate • Generally no external utilities required • Disadvantages • No air removal • Can only sterilize non-volumetric items (tools)

9. Gravity Displacement • Uses steam (lighter than air) to force the air out of the chamber • No vacuum • No separate steam generator • Generally only good for solid, unwrapped items • Cannot remove air* • Air can “puddle” making cold spots in load • Can be used for liquid loads** Confidential

10. Positive Pressure Displacement • Similar to gravity displacement in principle, but steam is supplied by external steam generator • Advantages • Better for air removal since steam is pulsed in, disturbing air, which mixes and purges more chamber/load air out. • Disadvantages • Higher cost than gravity displacement autoclaves • Generally smaller chamber than gravity due to other components like the steam generator

11. Vacuum (Vacuum Displacement) • Vacuum pump removes air from the chamber • Vacuum is broken with steam • Advantages • Best for air removal • Can sterilize all types of hard goods, including bottles, cylinders, and other volumetric cont. • Disadvantages • Most expensive • Load items must be able to withstand vacuum

12. Standard Autoclave Cycles • Vacuum cycles • Used for porous or hard goods, • Volumetric containers • Removes air from loads • Gravity cycles • Used for liquid loads • No vacuum • Air overpressure • Used for small volume liquid (terminal sterilization) • Leak Test • Bowie-Dick, Daily Air Removal Test (DART)

13. Definition of Terms • Dwell/Exposure phase • Start of the actual sterilization cycle (dry goods) • All points in load are at or above 121°C • Prevac Pulse/Vacuum Pulse • Part of the heat-up phase in vacuum autoclaves where the chamber is put under vacuum then the vacuum is broken with steam • Load Probe • In some autoclaves, an internal probe used to control the liquid load cycle. Placed into the center of mass of an equal mass of water. Only used for liquids • Slow exhaust phase • In a liquid cycle following exposure, the chamber is kept under pressure until the liquid temperature is below 100°C to prevent the rapid boil-off of the liquids Confidential

14. Definitions (continued) • Clean Steam • Steam generated from a DI or Purified-quality source water • Pure Steam • Steam generated from a WFI-quality (pyrogen-free) source water • Plant Steam • Steam generated from a non-tested or city water-quality source water • Can be used as the heat source for the jacket since there is no product contact • Can be used as the heat source to generate clean steam • Jacket • surrounds autoclave chamber and is kept heated to a few degrees below sterilization temperature as an efficiency so that the sterilant is not also used as the heat source • Sterile • To make free from live bacteria or other microorganisms. • To render free of microorganisms: decontaminate, disinfect, sanitize. Confidential

15. Validation • What needs to be validated? • New equipment (IQ/OQ/PQ) • New load configurations (PQ) • Equipment with new major components (OQ,PQ) • Relocated equipment (IQ,OQ,PQ) • Equipment with new utilities (IQ, OQ) • …And how often? • Industry standard for autoclaves is yearly or • When one of the above conditions is true

16. Stages of validation • Design Validation • Not always required • Installation Qualification • Operational Qualification • Performance Qualification • A Cycle development phase should normally precede PQ to get the most out of the validation effort

17. Design Qualification • Should be done prior to purchase • Considers user requirements when deciding on the specific design of a system or equipment • Ideally, selects a suitable, preferred vendor for the appropriate system

18. Installation Qualification • Requires Protocol • Ensures that equipment is installed properly • Per manufacturer’s utility requirements • Per manufacturer’s space requirements* • Identification and verification of all system elements, parts, services, controls, gauges and other components

19. Operational Qualification • Requires protocol • Tests alarms • Tests unit at all high and low limits • Worst case conditions • Tests software limits • Very intensive testing • Requires draft of SOP at this point • Must be approved prior to start of PQ

20. Cycle Development • Should be done prior to PQ • Uses thermocouples (TCs) and a datalogger • Figures out worst-case load configuration • Finds best parameters/set points to use • Lets you know if your autoclave will sterilize your goods • Finds minimum load • Find any other autoclave limitations

21. Planning a Load Configuration • Some things to consider when planning your load: • How many do you need at once? How often? • How many do you have? • How many will fit? Is it packed too tightly? • Does your autoclave have the capacity? • Do you need to reallocate/re-aliquot into smaller containers? • Is efficiency worth failure? (no) • Should you individual wrap/package or can you put several items in the same pouch? • Is it overpackaged? Confidential

22. Performance Qualification • Shows consistency of performance over time • 3x Empty Chamber per cycle type • Validates the established “worst-case” load from cycle development runs • Also can have a minimum load* (generally just for liquid cycles) • Requires a worst-case load per cycle type • Liquids • Hard goods, etc. • Load configurations must be documented in SOP

23. Lethality and Sterility Assurance Level • Lethality, or F0, is a mathematical representation of the amount of kill per unit time • At 121.1°C, you get 1.0 F0 per min, or 1 log reduction in a theoretical population of organisms • The industry standard Sterility Assurance Level (SAL) target is 10-6, which requires a 12-log reduction to attain • If you get 12 F0 during your exposure, then you effectively have a 1 in a million chance of contamination. • F0 Formula: 10^((temp-121.1)/10) (calculated by Kaye or manually) • FH Formula: 10^((temp-170.0)/20) (calculated by Kaye or manually) • SAL Calculation: LOG B = LOG A - (F0 /D121) • Log B = SAL, Log A= Spore population, F0 = total for run, D121=1

24. CalculatingLethality • Notated as F0for steam sterilization or • The equivalent sterilization time spent at the base temperature, which is 121°C • Lethality Rate : 10 (T-Tb)/Z • F0= Δt x Lethality Rate • Δt : Cycle time • T : Actual Cycle temperature • Tb : Base Temperature • Z : Microbial Death Rate Constant Confidential

25. Thermal resistance of microbes • What is the D-Value? (decimal reduction value) • The D-Value is the time, in minutes, at a specific temperature, required to reduce the surviving microbial population by 1-log (90%). Confidential

26. What is the Z-value? • The Z-value is the temperature change required to result in a 1-log reduction in D-value • To determine Z-value, one must determine the D-value at 3 different temperatures then plot the value. Confidential

27. Lethality Table Confidential

28. Biological Indicators • PQ uses biological indicators to prove efficacy • Besides Lethality and Sterility Assurance Level, there is a necessity to show a PHYSICAL kill, which is achieved using Biological Indicators. • 106population of Geobacillusstearothermophilusis generally used due to its robustness • Ampoule BIs are used for liquid loads • Spore strips are used for hard goods loads • Placed in “cold spots” found during cycle development adjacent to thermocouples • **Ampoule BIs CANNOT be used for dry goods, and strips CANNOT be used for liquid loads**

29. More about Biological Indicators • Spore Strip BIs require a separate growth medium • Strips must be placed in the growth medium using aseptic technique and incubated at 55-60°C in a validated incubator. • Ampoule BIs are self-contained and need only be placed into incubator. If they are contaminated, they will become turbid and the medium will change color • BIs must be properly stored and should be inventory controlled. • Some applications use different organisms other than Geobacillusstearothermophilus, but autoclaves use this organism due to its thermal robustness • Spore suspension indicators must be sterile filtered, plated, then counted to measure any viable growth Confidential

30. Other Species of BIs - overview • Bacillus pumilus is used for gamma irradiation and e-Beam due to its resistance to radiation • Bacillus subtilus is used for EtOH, H2O2, and Dry heat sterilization due to its resistance to alkylating agents, oxidizing chemicals, and heat. • Aspergillus niger is used for UV sterilization due to its Ultraviolet resistance • NOTE: depending on the application, these will be spore suspensions of various populations, not strips OR ampoules Confidential

31. Revalidation • Performed Annually on maximum and minimum load (no exceptions) • Performed if repairs done on equipment • (change control assessment) • Performed if load configuration is changed • Investigative runs (PQ addenda) can be performed here in lieu of full revalidation

32. Safety (General) • High temperatures! • Autoclaves operate at very high temperatures • DO NOT reach into an autoclave with bare hands • Jacket is usually heated and will burn you • Always wear proper PPE, minimally high temperature gloves Confidential

33. Safety (Liquids) • Wear glasses or face shields for liquid loads as liquids can splatter • Allow sufficient cool down time after removing load, prior to handling TCs • Ensure that any caps are loose prior to loading autoclave • Allow liquids to cool prior to removing Biological Indicators Confidential

34. Questions? Confidential

35. You Made It! Thank you for your attention! Call me at 303-884-8175 or stop me anytime if you think of anything later Confidential