BASIC PRINCIPLES IN THE VALIDATION OF STERILE PRODUCTS Theoretical Approaches Generally, five basic steps are necessary to validate
any manufacturing process: 1. Written documentation 2. Manufacturing parameters 3. Testing parameters 4. In-process controls 5. Final product testing In sterile product manufacturing, five major steps are involved in approaching the validation of a sterile process. These are outlined below using thermal sterilization as the example process. 1. Select or define the desired attributes of the product. Example: The product will be sterile. 2. Determine specifications for the desired attributes. Example: The product will be sterilized by a sterilization process sufficient to produce a probability of nonsterility of one out of 1 million containers (10−6). 3. Select the appropriate processes and equipment. Example: Use microbial kinetic equations such as Eq. (11) to determine the probability of nonsterility. Select cleaning equipment and container component procedures designed and validated to reduce the product bioburden to the lowest practical level. Select an autoclave that can be validated in terms of correct operation of all mechanical controls. Use the appropriate types of thermocouples, thermal sensing devices, biological indicators, integrated chemical indicators, and culture media to conduct the validation tests. 4. Develop and conduct tests that evaluate and monitor the processes, equipment, and personnel. Examples: a. Determine microbial load counts prior to container filling. b. Determine D and Z values of biological indicator organism. c. Perform heat distribution studies of empty and loaded autoclave. d. Perform heat penetration studies of product at various locations in the batch. 5. Examine the test procedures themselves to ensure their accuracy and reliability. Examples: a. Accuracy of thermocouples as a function of variances in time and temperature. b. Repeatability of the autoclave cycle in terms of temperature and F value consistency. c. A challenge of the sterilization cycle with varying levels of bioindicator organisms. d. Reliability of cleaning processes to produce consistent low-level product bioburdens. Each validation process should have a documented protocol of the steps to follow and the data to collect during the experimentation. As an example, App. I presents a protocol for the validation of a steam sterilization process.Upon completion of the experimental
particularly in the sterilization of barrier isolators. The interval between validation studies strictly depends on the judgment of the validation team based on the experience and history of the consistency of the process. Gaseous validation and radiation validation approaches will be focused on ethylene oxide and gamma radiation. documentation should present original validation records. Validation approaches and procedures used for most of these methods will be addressed in the remainder of this chapter. light. and filtration. and data from the revalidation studies. Some extra coverage will be given to vapor phase hydrogen peroxide because of its increased application. Table 3 lists the sterilization methods used for sterile products. generally will follow the same principles as those discussed for ethylene oxide and gamma radiation. The other gaseous and radiation methods. The first four methods destroy microbial life. however. it must be controlled to assure that the process consistently produces a product within the specifications established by the validation studies.phase of validation. Once a process has been validated. radiation. while filtration removes micro-organisms. an example of which is shown in Table 2. There are five basic methods—heat. respectively. As shown in Table 2. gas.
. a schedule of revalidation dates. The results may be summarized on a summary sheet. the data are compiled and evaluated by qualified scientific personnel.
and filtration. 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.”
Moist Heat Sterilization Definition D-value The time in minutes required for a one-log or 90% reduction of a specific microbial population under specified lethal conditions. that destroys or eliminates all viable microbes including resistant bacterial spores from a fluid or a solid. calculated using a specific value of z.VALIDATION OF STERILIZATION JM Tech. Fo value(accumulated Fo) The term "Fo " is defined as the number of equivalent minutes of steam sterilization at temperature 121. flushing with a sterilizing solution such as hydrogen peroxide (H2O2) or ozone (O3). Do-Young Ahn Moist Heat Sterilization Definition Sterilization “The act or process. dry heat at 230℃.” Examples of sterilization methods are : steam treatment at 121℃. Sterility “The reduction of anticipated levels of contamination in a load to the point where the probability of survival is less than 10-6. irradiation. physical or chemical. F(T.
Moist Heat Sterilization Definition F value(lethal rate. instantaneous Fo) The F value is a measurement of sterilization effectiveness. Fo = ♣ 10^((121-T)/z)*′ t
Moist Heat Sterilization
.z) is defined as the equivalent time at temperature T delivered to a container or unit of product for the purpose of sterilization.1°C delivered to a container or unit of product calculated using a z-value of 10°C.
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.
Moist Heat Sterilization Methodology Bioburden Sterilization Provides a probability of survival of less than 1 in 106 for the most resistance bioburden expected in the load. Requires ongoing monitoring or control over bioburden. Requires information on the numbers and heat resistance of the BI. BI may not be inactivated Requires information on the numbers and heat resistance of the BI.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
.Methodology Overkill Sterilization Provides a minimum 12 log reduction of a resistant BI w/ a known D-value of not less than 1 minute.2 g/ℓ Density of steam at 100℃ = 0.
Moist Heat Sterilization Sterilizer Cycle Gravity Displacement Difference of density Density of air at 20℃ = 1. Requires ongoing monitoring or control over bioburden.
Moist Heat Sterilization Sterilizer Cycle Prevacuum cycle A more effective method By means of a mechanical vacuum pump or a steam eductor Vacuum as low as 15~20 mmHg. BIER(biological indicator evaluator resistometer) Inoculate the spore into the actual solutions For solid materials. apply for 8~10 min. 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. 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. precut strips
Moist Heat Sterilization Preparing for Validation Temperature sensing devices :
. raining effect by external pump w/ cooling
Moist Heat Sterilization Cycle Development Consider factors into account Nature of the load : porous materials.
130 ℃ Correction factors Stability : ♣0. HTR Calibration of thermocouples At two temperatures : 0 ℃.5℃♣
Moist Heat Sterilization Preparing for Validation Autoclave Validation nozzle and adaptor Data logger : digital output and multi-channel device BIs or biological challenges Loads Moist Heat Sterilization 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
Moist Heat Sterilization Validation Protocol A description of the following studies
. 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.03℃ Accuracy : 0.
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. and min. sizes and fill volume to be validated The number of the thermocouples used depends on the container volume
. normally 15~20 probes Moist Heat Sterilization Heat Distribution Studies At loaded chamber heat distribution test.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 Moist Heat Sterilization 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. adjacent to the control sensor At least 10 probes. 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 Moist Heat Sterilization Container Mapping To determine the coolest point within the liquid filled container Temperature mapping should be conducted on all the different container types.
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
Moist Heat Sterilization 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
Moist Heat Sterilization 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.Possible to use a single thermocouple at different positions. coli Type of BI : Spore strips or spore suspension into the suspending medium
. and can be conducted in a smaller autoclave or retort Penetration thermocouples should be positioned at the cold spot having lowest temperature or Fo
Moist Heat Sterilization 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.
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.
.Microbial challenge studies are conducted concurrently with the heat penetration studies Moist Heat Sterilization 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 Moist Heat Sterilization 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 Moist Heat Sterilization Controversial Issues Incubation of the sterility test : 7 days vs. Z=(T2-T1)/Log(D2/D1). stearothermophilus as a BI Z-value measures the rate of change in the D-value as a function of temperature.
D 값을 1/10 로 단축시키는데 소요되는 온도상승값 Temperature uniformity of the heat distribution studies may be influenced by Type Size Design Installation of the sterilizer Container mapping studies can be conducted in a small autoclave or retort. The temperature profile of the container should remain constant among different sterilizing chamber.