Regulatory Requirement of Sterilization Process Validation

1.INTRODUCTION (1,2,3,4,6,7)

2009

Validation is an integral part of quality assurance and its simple meaning is action of proving. It involves controlling the critical steps of a system, which result in output of repeatable attributes. In validation all afford done to remove all the variable those affected the quality of product or process. A validation of process demonstrate that when a process is operated with in specified limit, it will consistently produce product complying with predetermined (design) requirements. The foremost priority of regulatory agencies is to ensure the safety of public, efficiency of process & quality of product. Behind the regulation of any process the primer basis is that to ensure the minimize the health hazard & assurance of Health of public. In all over the world various health agencies of govt. of various countries & cumulative agencies like who are take action to regulate the process of manufacturing of drug of their distribution in all over world. These agencies also provide the guide line to validation of process & equipment to minimize the variable who affected the product quality. In USA the US FDA (US Food & Drug Administrations) regulated the manufacturing & Distribution of Drug According to US FDA the prerequisites of validation are as given 21 CFR 211.110 : Validation of performance of manufacturing process. 21 CFR 211.100 : Written procedure for production & process control. 21 CFR 211.113 : Validation of Any Sterilization process Following section of CGMP under section 21 CFR 211 refer to validation : 211.68 : Validation of Computerized & automated process 211.113 (b) : Validation of sterilization processes Prerequisites for Validation (2) : Process Development [21 CFR 820.30 Design Control] Process Documentation [21 CFR 211 Sub Part F & Sub Part J Record & Report] Equipment qualification [21 CFR 211 Sub Part C] Calibration [21 CFR 211 Sub Part C]
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Regulatory Requirement of Sterilization Process Validation

Analytical method [21 CFR 211 Sub Part I] Equipment cleaning & maintenance [21 CFR 211.67] Change control [21 CFR 211.100]

2009

CGMPS, Human & Veterinary Drugs was published in September 1978. The regulation are published in the CFR Title 21 Part 210 & 211. In the regulation the term validation is not defined & only mention in only in four sections. The specific section are related to computer data validation, COA data validation, sterilization process validation & Analytical method validation. In 1987 the FDA published the guideline on sterile drug products produced by Aseptic processing. Failure to validate is a term encountered in FDA form 483. Three principles are involved in the validation process for sterile product. 1. 2. To built sterility in to product. To demonstrated to a certain maximum level of provability that the processing & sterilization method have established sterility to all unit of a product batch. 3. To provide greater assurance & support of results of the end product sterility testing.

Regulatory Requirement of Sterilization Process Validation

2009

2. GUIDANCE FOR INDUSTRY FOR THE SUBMISSION OF DOCUMENTATION FOR STERILIZATION VALIDATION 2.1 INTRODUCTION A. Purpose This document is intended to provide guidance for the submission of information and data in support of the efficacy of sterilization processes in drug applications for both human and veterinary drugs. The recommendations in the guidance apply to applications for sterile drug products (new drug applications, new animal drug applications, abbreviated new drug applications, abbreviated antibiotic applications, and abbreviated new animal drug applications). These recommendations also apply to previously approved applications when supplements associated with the sterile processing of approved drugs are submitted. Information and data in support of sterility assurance may also be necessary in investigational new drug and investigational new animal drug applications. In the Federal register of October 11, 1991 (56 FR 51354), the agency published a proposed rule entitled "Use of Aseptic Processing and Terminal Sterilization in the Preparation of Sterile Pharmaceuticals for Human and Veterinary Use." This guidance is not a substitution for or a supplement to that proposed rule. Regardless of whether the applicant uses terminal sterilization or aseptic processing to manufacture a drug product that is purported to be sterile, certain information about the validation of that process should be submitted for both of those types of sterilization. B. Documenting Sterilization Process Validation The efficacy of a given sterilization process for a specific drug product is evaluated on the basis of a series of protocols and scientific experiments designed to demonstrate that the sterilization process and associated control procedures can reproducibly deliver a sterile product. Data derived from experiments and control procedures allow conclusions to be drawn about the probability of nonsteirile product units (sterility assurance level). Based on the scientific validity of the protocols and methods, as well as on the scientific validity of the results and conclusions, the agency concludes that the efficacy of the sterilization process is validated. Whether a drug product is sterilized by a terminal sterilization process or by an aseptic filling process, the efficacy of the sterilization process may be validated
3

Regulatory Requirement of Sterilization Process Validation

2009

without the manufacture of three production batches. Sterilization process validation data, however, should be generated using procedures and conditions that are fully representative and descriptive of the procedures and conditions proposed for manufacture of the product in the application The Center for Drug Evaluation and Research's (CDER's) and the Center for Veterinary Medicine's (CVM's) review of the validation of the sterilization process consists of a scientific evaluation of the studies submitted in the applications. This review is conducted by FDA's review staff, and is part of a cooperative effort between the review staff, compliance staff, and field investigators to ensure the overall state of control of the sterile processing of human and veterinary drug products. Information and data in support of sterility assurance may be provided directly to the application or by specific reference to a drug master file (DMF), a veterinary master file (VMF), or another application. Letters of authorization to refer to the referenced files should be included. C. Remarks This guidance is intended to provide recommendations for the types of information applicants should include in human and animal drug applications. Regulatory requirements for the submission of information and data in various applications are specified in the sections listed below: 1. Human Drugs: Investigational new drug applications New drug applications Abbreviated new drug and abbreviated antibiotic drug applications Supplements to NDA's and ANDA's 2. Animal Drugs: Investigational new animal drug applications New animal drug applications 21 CFR Part 511 21 CFR 514.1
4

21 CFR 312.23(a)(7) 21 CFR 314.50

21 CFR 314.94 and 314.50 21 CFR 314.70

Regulatory Requirement of Sterilization Process Validation

Supplements to NADA's 21 CFR 514.8

2009

2.2 INFORMATION FOR TERMINAL MOIST HEAT STERILIZATION PROCESSES The following types of information should be submitted in support of sterility assurance for products produced using terminal moist heat sterilization. Although the following outline directly addresses moist heat processes, the same types of information would generally pertain to other terminal sterilization processes (e.g., ethylene oxide or radiation). (See section III of this guidance.) The following information should be submitted for each facility to be used in the manufacture of the proposed drug product: A. Description of the Process and Product 1. The Drug Product and Container-Closure System Descriptions of the drug product and the container-closure system(s) to be sterilized (e.g., size(s), fill volume, or secondary packaging). 2. The Sterilization Process A description of the sterilization process used to sterilize the drug product in its final container-closure system, as well as a description of any other sterilization process(es) used to sterilize delivery sets, components, packaging, bulk drug substance or bulk product, and related items. Information and data in support of the efficacy of these processes should also be submitted. (See also sections II.B. and II.C. of this guidance.) 3. The Autoclave Process and Performance Specifications A description of the autoclave process, including pertinent information such as cycle type (e.g., saturated steam, water immersion, and water spray), cycle parameters and performance specifications including temperature, pressure& time and minimum and maximum Fo . Identify the autoclave(s) to be used for production sterilization, including manufacturer and model. 4. Autoclave Loading Patterns
5

Regulatory Requirement of Sterilization Process Validation

A description of representative autoclave loading patterns should be provided. 5. Methods and Controls to Monitor Production Cycles

2009

Methods and controls used to monitor routine production cycles (e.g., thermocouples, pilot bottles, and biological indicators) should be described, including the number and location of each as well as acceptance and rejection specifications. 6. Requalification of Production Autoclaves A description of the program for routine and unscheduled requalification of production autoclaves, including frequency, should be provided. 7. Reprocessing A description and validation summary of any program that provides for reprocessing (e.g., additional thermal processing) of product should be provided. Please note that the stability program is also affected by additional thermal processing. For further information

concerning the stability program, reference is made to the Center for Drug Evaluation and Research "Guideline for Submitting Documentation for the Stability of Human Drugs and Biologics" and to the Center for Veterinary Medicine "Drug Stability Guideline." B. Thermal Qualification of the Cycle 1. Heat Distribution and Penetration Studies Heat distribution and penetration study protocols and data summaries that demonstrate the uniformity, reproducibility, and conformance to specifications of the production sterilization cycle should be provided. Results from a minimum of three consecutive, successful cycles should be provided to ensure that the results are consistent and meaningful. 2. Thermal Monitors The number of thermal monitors used and their location in the chamber should be described. A diagram is helpful. 3. The Effects of Loading on Thermal Input

Regulatory Requirement of Sterilization Process Validation

2009

Data should be generated with minimum and maximum load to demonstrate the effects of loading on thermal input to product. Additional studies may be necessary if different fill volumes are used in the same container line. Data summaries are acceptable for these purposes. A summary should consist of, for example, high and low temperatures (range), average temperature during the dwell period, minimum and maximum Fo values, dwell time, run date and time, and identification of the autoclave(s) used. These data should have been generated from studies carried out in production autoclave(s) that will be used for sterilization of the product that is the subject of the application. 4. Information Included in the Batch Record The batch record supplied with the chemistry, manufacturing, and controls section of the application should identify the validated processes to be used for sterilization and for depyrogenation of any container-closure components. This information can be included in the batch record by reference to the validation protocol or standard operating procedure (SOP). Validation information should be provided as described above. C. Microbiological Efficacy of the Cycle Validation studies that demonstrate the efficacy (lethality) of the production cycle should be provided. A sterility assurance of 10-6 or better should be demonstrated for any terminal sterilization process. This level of sterility assurance should be demonstrated for all parts of the drug product (including the container and closure, if applicable), which are claimed to be sterile. The specific type of study and the methods used to carry out the study (or studies) are product and process specific and may vary from manufacturer to manufacturer. In general, the following types of information and data should be provided. 1. Identification and Characterization of Bioburden Organisms Describe the methods and results from studies used to identify and characterize bioburden organisms. The amount and type of information supplied may be dependent on the

validation strategy chosen. For example, more information may be needed for bioburdenbased autoclave processes than for overkill processes. Information concerning the number, type, and resistance of bioburden organisms may be necessary, including those organisms associated with the product solution and the container and closure. It may be necessary to identify the most heat- resistant bioburden organisms.
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Regulatory Requirement of Sterilization Process Validation

2. Specifications for Bioburden

2009

Specifications (alert and action levels) for bioburden should be provided. A description should be included of the program for routinely monitoring bioburden to ensure that validated and established limits are not exceeded (e.g., frequency of analysis and methods used in bioburden screening). The methods provided should be specific. 3. Identification, Resistance, and Stability of Biological Indicators Information and data concerning the identification, resistance (D and Z values), and stability of biological indicators used in the biological validation of the cycle should be provided. If biological indicators are purchased from a commercial source, it may be necessary to corroborate the microbial count and resistance, and provide performance specifications. 4. The Resistance of the Biological Indicator Relative to That of Bioburden Studies characterizing the resistance of the biological indicator relative to that of bioburden may be necessary. Resistance in or on the product (i.e., in the product solution, or on the surface of container or closure parts or interfaces) should be determined as necessary. If spore carriers are used (e.g., spore strips), the resistance of spores on the carrier relative to that of directly inoculated product should be determined, if necessary. 5. Microbiological Challenge Studies Microbiological validation studies should be submitted that demonstrate the efficacy of the minimum cycle to provide a sterility assurance of 10 or better to the product under the most difficult to -6 sterilize conditions (e.g., the most difficult to sterilize load with biological indicators at microbiological master sites or in master product or both). Use of a

microbiological master product or site should be supported by scientific data. Microbiological master sites or solutions are those sites or solutions in which it is most difficult to kill the biological indicator under sterilization cycles that simulate production conditions. D. Microbiological Monitoring of the Environment Section 211.160 of the Code of Federal Regulations requires, in part, the establishment of scientifically sound and appropriate specifications, standards, sampling plans, and test procedures designed to ensure that components, drug product containers, closures, in8

Regulatory Requirement of Sterilization Process Validation

2009

process materials, and drug products conform to appropriate quality standards. Therefore, a microbiological monitoring program for production areas along with a bioburden monitoring program for product components and process water should be established. Process water includes autoclave cooling water. Applicants should provide information concerning this program. Frequency, methods used, action levels, and data summaries should be included. A description of the actions taken when specifications are exceeded should be provided. E. Container-Closure and Package Integrity An applicant should provide scientific validation studies (and data) in support of the microbial integrity of the drug packaging components. The following types of information should be included: 1. Simulation of the Stresses from Processing Experimental designs should simulate the stresses of the sterilization process, handling, and storage of the drug and their effects on the container-closure system. Physical, chemical, and microbiological challenge studies may be necessary. 2. Demonstrate Integrity Following the Maximum Exposure Container-closure integrity should be demonstrated on product units that have been exposed to the maximum sterilization cycle(s). If a product is exposed to more than one process, then exposure to the maximum cycle of all processes should be incorporated into the study design. 3. Multiple Barriers Each barrier that separates areas of the drug product claimed to be sterile should be separately evaluated and validated. 4. The Sensitivity of the Test The sensitivity of the experimental method used for container-closure integrity testing should be specified and provided. 5. Integrity over the Product Shelf Life

Regulatory Requirement of Sterilization Process Validation

2009

Microbial integrity of the container-closure system should be demonstrated over the shelf life of the product. (See section V.A. of this guidance.) F. Bacterial Endotoxins Test and Method The bacterial Endotoxins test used for the product should be described. The description should include qualification of the laboratory, inhibition and enhancement testing and results, determination of noninhibitory concentration and maximum valid dilution. For further information see the agency guidance entitled "Guideline on Validation of the Limulus Amebocyte Lysate Test As an End-Product Endotoxin Test for Human And Animal Parenteral Drugs, Biological Products, and Medical Devices." G. Sterility Testing Methods and Release Criteria Sterility test methods should be described and should include the protocol for the selection of representative units during production. When test methods differ significantly from compendial test methods, a demonstration of the equivalency to the compendial method should be provided. Testing performed within barrier systems should be described, and information concerning validation of the barrier system may be necessary. H. Evidence of Formal, Written Procedures Section 211.113(b) of the Code of Federal Regulations requires that written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, be established and followed. Such procedures should include validation of any sterilization process. Therefore, evidence should be provided that there are formal, written procedures describing the elements listed above and that these procedures are followed. Such evidence may consist of SOP's, listing of SOP's, and protocols submitted as part of these elements. 2.3 OTHER TERMINAL STERILIZATION PROCESSES Although the information above (sections I.A. through I.G. of this guidance) directly addresses moist heat processes, the same type of information would pertain to other terminal sterilization processes used singly or in combination to sterilize a drug product. The types of information outlined are, in general, also applicable to ethylene oxide and radiation (gamma and electron beam). These other processes should be addressed as each applies to the drug product, sterile packaging and in-process sterilization of components.
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Regulatory Requirement of Sterilization Process Validation

2009

Examples of such information might include: descriptions of loading configurations; qualification and validation of master load configurations; determination and validation of the efficacy of the minimum cycle to provide sterility assurance at the product master sites; requalification of the cycle; provisions for resterilization. Specifications and monitoring program for product bioburden and container-closure integrity. Specific examples are provided below to demonstrate the application of these concepts to other sterilization processes. Additional information relating to the effects of the sterilization process on the chemical and physical attributes of the drug substance or drug product may be applicable, and should be supplied to the chemistry, manufacturing, and controls section of the application. A. Ethylene Oxide 1. Description of the Sterilizer The sterilizer(s) and controlled site(s) for prehumidification and aeration of the product load should be described. 2. Cycle Parameters The parameters and limits for all phases of the cycle, e.g.,prehumidification, gas concentration, vacuum and gas pressure cycles, exposure time and temperature, humidity, degassing, aeration, and determination of residuals should be specified. Specific procedures used to monitor and control routine production cycles to assure that performance is within validated limits should be provided. 3. Microbiological Methods The microbiological methods (growth medium, incubation temperature, and time interval) for cultivating spores from inoculated samples during validation experiments should be described as well as the microbiological methods used as part of routine production cycles. 4. Stability The program for monitoring the stability of packaging and the integrity of the containerclosure system barrier over the claimed shelf life should be described.

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Regulatory Requirement of Sterilization Process Validation

B. Radiation 1. The Facility and the Process

2009

The radiation facility should be identified. The radiation source, method of exposure (i.e., movement through the irradiator), and the type and location of dosimeters used to monitor routine production loads should be described. If the low dose site is not used for routine monitoring, data that show the dose relationship between the two sites should be provided. 2. The Packaging of the Product The packaging of the drug product within the shipping carton and within the carrier should be described. 3. Multiple-Dose Mapping Studies Multiple-dose mapping studies for identification of low and high dose sites and demonstration of uniformity and reproducibility of the process should be described. 4. Microbiological Methods and Controls The microbiological methods and controls used to establish validate, and audit the efficacy of the cycle should be described. 5. Monitoring Stability The program for monitoring the stability of packaging and the integrity of the containerclosure system barrier over the claimed shelf life should be described.

12

Regulatory Requirement of Sterilization Process Validation

3. AUTOCLAVE VALIDATION PROTOCOL (9,3,2,16) Validation of the Autoclave is classified into the following DQ- Design qualification IQ- Installation qualification OQ Operational Qualification PQ Performance Qualification

2009

The validation is being taken up to cater to the new requirements of the GMP. Since it is already in use only OQ and PQ will be considered.

VALIDATION TEAM: NAME

TABLE-1 ROLE & RESPONSIBILITY SIGN.

DEPARTMENT DESIGNATION

Q.C Q.C

microbiologist Manager microbiology

Prep ration Protocol checking (documentation of result) Co-ordination & checking Utility support approval

Q.C maintenance Q.C

manager manager General manager

Q.A

Sr. manager

authorization

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Regulatory Requirement of Sterilization Process Validation

OPERATIONAL QUALIFICATION PROTOCOL (OQ) PURPOSE :

2009

To demonstrate and document that the operations of the Autoclave take place as specified . SCOPE : Autoclave xxxxx will be qualified to meet OQ. RESPONSIBILITY : Microbiologist, Manager Q.C PROCEDURE : This should be performed by external agency like IIME. Verify the following as per instrument operating procedure and calibration certificate kept in place before validation. Temperature display on autoclave. Compound pressure gauge of Autoclave. Acceptance Criteria : All calibration data found to be within the acceptable norms of calibration certificate. Calibration of Thermocouples : Calibrate all the thermocouples of data logger before and after the validation using standard thermometer and also made available partys calibration certificates. Acceptance criteria: The variation between the temperature of thermocouples and the standard thermometer found to be within the acceptance criteria. Heat Distribution Studies Carry out heat distribution studies by using a multi-point data logger and maintain holding time for 15 minutes at 15 lbs. by fixing all the 12 probes as per diagram-1. Record the temperature and lag time of each probe as per Annexure 1 & 2. Acceptance criteria All probes must reach temperature 121-124C and pressure must be within 15 to 18 lbs for 15min cycle.

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Regulatory Requirement of Sterilization Process Validation

12 9 11 10

2009

8 4

6 1

5 3

7 2

FIG-1: Probe No.1 to 12 inside the chamber

Load Pattern
Maximum Load Load with all the glassware and media filled upto 70%, of the chamber and the details are as follows. Test-1 : 250ml Conical flasks = 12 Nos with media, 13 Nos without media, 500ml Conical flasks with media = 4nos, 1000ml Conical flasks with media =4nos, Pipette10ml=10nos,Pipette 2ml=10nos, Pipette 5ml=10nos, Pipette 1ml = 10nos,100ml bottles=20nos ,Filtering unit=10nos, Test tubes =25nos Minimum Load Load with all the glassware and media required for a days analysis (average). 250ml Conical flasks with media = 6nos, 500ml Conical flask with media 3 nos., 1000ml Conical flask - 1 Pipette 10ml= 10nos, Pipette 2ml= 10nos, Pipette 5ml= 10nos, Pipette 1ml=10nos, Bottles=10nos, Test tubes - 25nos. Record the temperature and lag time and tabulate as per annexure 1.

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Regulatory Requirement of Sterilization Process Validation

Test name: time TABLE-2 THERMOCOUPLE CALIBRATION Probe temp(oc) 1 2 3 4 5 6 7 8 9 10 11 12 Display temp(0c) pressure (psi/lb2)

2009

Log time

PERFORMANCE QUALIFICATION PROTOCOL (PQ) PURPOSE : To provide a performance qualification protocol for Autoclave. SCOPE : Specified to Autoclave xxxxx. RESPONSIBILITY : Microbiologist, Manager Q.C PROCEDURE : Heat Penetration Studies Carry out the heat penetration studies by using a multi point data logger for the following loads mentioned. Record the temperature and lag time if any as per annexure 1 and 2. Acceptance criteria: All 12 probes must reach temperature 121C to 124C and pressure must be within 15 to 18 lbs. for 15 min cycle. Microbial limit test : Incubate the sterilized media flask or tubes from any one Maximum and Minimum load of heat penetration studies and observe for nutritive properties. Bacteria: 30 35 C for 72 hrs, Fungi : 20 25C for 120 hrs

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Regulatory Requirement of Sterilization Process Validation

Acceptance criteria:

2009

No microbial growth should be observed i.e. Negative control and Nutritive properties of media must pass Microbial challenge test : Keep ampoules containing spores suspension of Bacillus stearothermophilus 106 population at various location of the autoclave along with probes and maintain the sterilization temperature at 15psi and 121C during the heat penetration studies, once on the maximum load. Acceptance criteria: Autoclaved ampoules containing Bacillus stearothermophilus spores suspension ampoules should not show any colour change after seven days of incubation.

TABLE-3 :TESTS INVOLVED IN AUTOCLAVE VALIDATION TESTS Heat distribution Minimum load: test-1 Maximum load:test-1 Maximum load:test-2 Maximum load:test-3 LOAD PATTERN Empty load:test-1 All 12 probes must reach temp121oc to 124oc & pressure must be with in 15 -18 lbs for 15 min. cycle CRITARIA

Heat penitration

Minimum load: test-1 Minimum load: test-2 Minimum load: test-3 All 12 probes must reach temp121oc to 124oc & pressure must be with in 15 -18 lbs for 15 min. cycle

Maximum load:test-1
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Regulatory Requirement of Sterilization Process Validation

Maximum load:test-2 Maximum load:test-3

2009

Microbial limit tests

Maximum /minimum load

Negative control & nutritive properties must pass

Microbial challenge test

Maximum load

Autoclaved bacillus stearothermophilus spores suspension containing ampoules should not show any colour change after 7 days of incubation at 550c

DOCUMENTATION : Master Instrument used for validation of autoclave Institutes name and address carrying out calibration. Standard calibrating instrument name and number. Instrument certified against (Instrument of national or international standards) Date of calibration and validity period of calibration. Training certificate of persons (External agency) carrying out validation. Autoclave being calibrated : All temperature readings for autoclave being validated should be collected from the approved external agency like IIME. Validation report with observed any error, statement of calibration and next validation FREQUENCY : Once in a year until and unless no change in autoclave. In case of any change, the autoclave must be revalidated

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Regulatory Requirement of Sterilization Process Validation

Test name: time TABLE-4 THERMOCOUPLE CALIBRATION Probe temp (oc) 1 2 3 4 5 6 7 8 9 10 11 12 Display temp(0c) pressure (psi/lb2)

2009

Log time

TABLE-5 TESTS INVOLVED IN AUTOCLAVE VALIDATION TESTS Heat distribution LOAD PATTERN Empty load:test-1 All 12 probes must reach temp121oc to 124oc & pressure must be with in 15 -18 lbs for 15 min. cycle CRITARIA

Minimum load: test-1 Maximum load:test-1 Maximum load:test-2 Maximum load:test-3

Heat penitration

Minimum load: test-1 Minimum load: test-2 Minimum load: test-3 Maximum load:test-1 All 12 probes must reach temp121oc to 124oc & pressure must be with in 15 -18 lbs for 15 min. cycle

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Regulatory Requirement of Sterilization Process Validation

Maximum load:test-2 Maximum load:test-3

2009

Microbial limit tests

Maximum /minimum load

Negative control & nutritive properties must pass

Microbial challenge test

Maximum load

Autoclaved bacillus stearothermophilus spores suspension containing ampoules should not show any colour change after 7 days of incubation at 550c

CONCLUSION : Finally conclusion should be drawn based on the results of above tests and documented.

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Regulatory Requirement of Sterilization Process Validation

2009

4. D Value (2,3,8,9,10,11)
Microorganisms an exposure by Heat, chemical or radiation will die according to a logarithmic relationship between the concentration or population of living cell & the time exposure or radiation dose to the treatment. This relationship may be linear or Non linear. Thermal resistance of specific microorganisms is characterized by D value. Time required at a specific temp to reduce the surviving microbial population by 90% [one logarithmic reduction]. D value used to define the death of microorganism is the failure to reproduce when suitable condition for reproduction is provided. D term used to describe the relative resistance of a particular microorganism to a sterilization process. The D value time element is a critical parameter used in both the validation of a process as well as in the routine monitoring of validation process. It also defined as time required for a 90% reduction in microbial population e.g. (time/dose taken to reduce 1000 microbial cells to 100 cells) D value shown in mathematically form as :

U log N 0 log N u

equ..(1)

U = Exposure time or exposure dose N0 = initial microbial population (product bioburden) Nu = initial population after time u or dose unit Example: After 5 min. of product exposure to a temp of 121C the microbial population was reduced 2 105 to 6 103 then D value is :-

U log N 0 log N

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Regulatory Requirement of Sterilization Process Validation

D121 5 3.25 min log( 2 10 ) log(6 10 3 )
5

2009

Thus at 121C the microbial population is decrease by 90% every 3.28 min. Graphical representation of the semi logarithmic microbial death rate

Fig: 2: D Value graph

105 104 = 1 log Decrease 103 102 = 1 log Decrease For study of D value the microorganism spores are used as challenge microorganism. Because they are the most resistant microorganisms to wet & dry heat destruction. Most of the resistant microbiological spores known are found in two genera : Bacillus facultative anaerobe Clostridium strict anaerobe The most commonly used Heat resistant species are B.subtilus, G.stearothermophilus, Bacillus coagulans & C.sporogenes.Resustant Bactirial spores are commercially available as Biological indicator in following forms : Spore strips Spore dots
22

Regulatory Requirement of Sterilization Process Validation

Spore suspension

2009

Self contained unit containing spore strip & media in which they are to be incubated. A product being validated for sterility should be associated with a characteristic D value for microorganism. Value is independent of time when the response is logarithmic .

23

Regulatory Requirement of Sterilization Process Validation 5. Z value (Resistance Value)(2,3,10)

2009

D value determination is generally carried out under Isothermal Condition, but it has been proven experimentally that resistance of a microorganism may change with alteration in temp. So the study of change in rate of microbial inactivation with a change in temp. is known as z value. Z value is only relevant to the thermal sterilization process, the temp dependence of radiation of gas sterilization procedure is not widely defined in this manner. Z value is necessary component of calculations that allow comparison of spore lethality at different temp. The Z value is reciprocal of the slope resulting from the plot of the logarithm of the D value versus the temp at which the D value was obtained. So the Z value defines as temperature required for a one-log reduction in the D value.

Z value of 10C the accepted standard for steam sterilization of Bacillus sterothermophilus spore & for a Z value of 20C the purposed standard for Dry Heat Sterilization of Bacillus subtilis spores. Z value plot an important source for determination of D value of the indicator microorganism at any temp of interest. Magnitude of slope of fig. indicate the degree of lethality at temp is increased or decreased.

T2T1 log D1 log D2

Equ (2)

Z value is a necessary component of calculation that allow comparison of spore lethality at different temp.In the absence of alternative data the generally accepted Z-value assumption are :24

Regulatory Requirement of Sterilization Process Validation

2009

Steam sterilization Z = 10 C(18 F) Dry Heat sterilization Z = 20C (36 F)

25

Regulatory Requirement of Sterilization Process Validation 6. F Value / Lethality Value(2,3,4,10)

2009

It is expressed in minute. It is equivalent time at specific temperature delivered to a container or unit of product. F value centered at autoclave is called FO value & for Dry heat sterilizer (Depyrogenation) is called FH value. F value is used for measurement of sterilization effectiveness. The FO value is the number of equivalent minutes of steam sterilization at temp. 121C delivered to container of unit of product.

. TO = 121C for steam sterilization

Equ. (3)

TO = 170C for Dry Heat sterilization standard temp. According to US FDA steam sterilization process must be sufficient to produce an FO value at least 8 min. This means that coolest location in the sterilizer loading configuration must be exposed to an equivalent time of at least 8 min. of exposure to a temp. of at least 121C. F value is used as a measurement of sterilization effectiveness FO is defined as the number of equivalent minutes of steam sterilization at temp. 121.1C delivered to a container or unit of product calculating z value of 10C. For example if there is a stated FO value of 9. It is saying that the process being described is equivalent to exactly 9 min. at precisely 121.1C regardless of the process temp. & time used in cycle. The term FH is similar to FO & it used to describe the number of equivalent minutes of dry heat sterilization at temp. calculate using Z value of 20C. Mathematical FO In mathematical terms, FO is expressed as :

FO t 10 (T 121.1) / Z for steam sterilization

FO t 10 (T 170) / Z for Dry Heat sterilization

.t = time interval between measurements of T. T = temp. of sterilized product at time t.
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Regulatory Requirement of Sterilization Process Validation

2009

Z = Z value [10 C for steam sterilization & 20 C for Dry Heat sterilization] Exp 1 : For a process that run for 12 min. at 121.1C

FO t 10 (T 121.1) / Z FO 12 10121.1121.1 / 10

Z = 10 in steam sterilization

FO 12 10 FO 12 min

FO 12 10 (120.1121.1 / 10) FO 12 10 1 / 10
FO 12 10 0.10

FO 12 0.79 FO 9.48 min

Determination of minimum Required FO : FO amount can be determined by evaluation the desire level of sterility assurance required, together with the bioburden of the product being sterilized.
A B FO D121.1 (log 10 log10 )

Equ.. (4)

D121.1 = D value at 121.1C A = Bioburden per container B = Maximum acceptable sterility assurance level (SAL) Exp : The product being sterilized has a bioburden of 100 spores per container, D value at 121.1C is 3.3 min & Desire Acceptable sterilization limit is 106 So
A B FO D121.1C (log 10 log10 ) FO (3.3)[log 100 log106 ] 10

27

Regulatory Requirement of Sterilization Process Validation

FO (3.3)[ 2 [6]] FO 3.3 8 2.64 min

2009

So for sterilization process to achieve the desired destruction of 100 spores to the extent of a 10-6 SAL. FO relate the killing efficiency of the process at any temp to the killing effect produced at the desired sterilization temp of 121C.FO it used to describe the lethal effect upon microorganism at coolest location in the sterilizer, represent the degree of destruction of microorganism, & thus the safest condition for determining cycle time. Three factor affect the FO value :1. 2. 3. Container size, geometry & Heat transfer coefficient Product volume & viscosity Size and configuration of the batch load in sterilization

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Regulatory Requirement of Sterilization Process Validation

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7. Probability of Nonsterlity /Sterility Assurance Level (1,3,10)

Probability of nonsterlity defines the product free of microbial contamination probability of non-sterility for a sterilized product is 10-6 according to USFDA. Probability of non-sterility means after a equivalent time exposure period of FT unit, the microbial population having an initial value of A has been reduced to a final B value 10 6 in statistically it means that one out of 1 million (106) unit of product or one out of 106 microorganism theoretically in no sterile product after sterilization. Probability of nonsterlity extrapolated from the D value slope when plotting the log of the microbial population versus time at specific temp.

Fig:4 Survival curve Curve show the effect of decreasing the microbial load (A) from 106 to 102 on the time required to achieve a probability of nonsterlity of 106. It is desirable that B be as low possible. This may accomplished in one of two ways (i) (ii) Reducing the Bioburden of bulk product. Increase the equivalent exposure time.

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Regulatory Requirement of Sterilization Process Validation

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8. Sterilization Cycle(2,3,9,10,12)
The designation of a sterilization cycle & development of cycle depend upon product characteristic specially on heat stability & bioburden. According to stability and microbiological character the sterilization cycle are as follows : (i) (ii) (iii) Overkill cycle approach BI/Bioburden cycle approach Bioburden approach

A sterilization cycle means time required for sterilization of a product to produce a SAL of 106 effectively means that the microorganism that could be present (i.e. bioburden) are killed 7 an additional 6 log reduction safety factor has been provided. The following example consider the point of sterilization cycle :Bioburden (Worst case) = 134 CFU (colony forming unit) [worst case item :- item in the load which are the most difficult to sterilize (determined by steam penetration study)] Ist step :- To reduce the microbial population from 134 to 1 log (134) = 2.13 so a 2.13 log reduction is required to reduce the population from 134 to 1 IInd step :-Now applying an additional 6-log reduction [means 106 to 1] or [1 to 0.000001] of microbial population. Log [106] = 6 So here a 6 log reduction is required to reduce the population from 1 to 0.000001 Total log reduction a microbial reduction from 134 to 0.000001 = 2.13 + 6 = 8.13 Therefore to provide a SAL of 106 d

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Regulatory Requirement of Sterilization Process Validation

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Therefore to provide a SAL of 10% with a bioburden of 134 CFU required a sterilization cycle that provides an 8.13 10 g reduction at specific temp. Bioburden & BI/Bioburden cycle are predominantly utilized in the sterilization of liquid filled containers for the sterilization of laboratory/production media & Certain in process liquids. Over kill approach selected for Heat stable material like filters, contained closure, hoses, Filling part & other hard good (Which are not absorbed/adsorbed air/moisture or soft good. Objective of over kill cycle is to assure that level of assurance, regardless of the number & the heat resistance of the organism in the load. In over kill method High of value are generally used. This may be chosen to provide at least a 12 log reduction (106 to 10-6 ) of micro organism with a D value of one in. at 1210 C for assurance of sterility bioindicators used to conform sterilization during validation study. These bioindicators are most often strip of suspension containing form 104 to 107 spores of highly heat resistant organism. ( G. Stearothermophilus) ( Exp:-D Value of G Stearothermophilus is observed 1.5 to as high 4 min. So over kill cycle as high as Fo=12 x 4 =48 min for 12 log reduction to provide 10-6 NSU.) Bioburden of D Value approach For items that are heal sensitive & Can't withstand on overkill approach then dramatically shorter the sterilization cycle required. Exp:- If the bioburden is low ( e.g. 10 CFU) or even moderately resistant ( D Value =0.5) than an ideal 30 min over sill cycle at 1210 C replaced by an ideal cycle of 3.5 min ( 7 log x 0.5 min/log =3.5 min) or by reducing the temp ( exp 1120 c for 30 min).It may be a significant approach to reduce the time.

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Regulatory Requirement of Sterilization Process Validation

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9.Calibration of Thermocouples(2,3,6,7)
In all qualification & Process conformation study the ability to accurate measure temp. is critical. The most versatile Temperature sensing devices for validation are TCs ( Thermocouples) Premium grade of wise, accurate to as close as 0.1OC at 121 OC are recommended. These must calibrate against a temperature standard traceable to the NIST ( formerly National Bureau of Standard), British, Standard or an acceptable national standard. Thermocouples must be sufficiently durable for repeated use as temperature indicator in steam sterilization validation & monitoring. Accuracy of thermocouples should be +0.5OC an error of 0.1OC in temp. Measurement produces a 2.3 % error in FoValue. Any Thermocouples that senses a temp of more than 0.5OC away from the calibration temp bath should be discarded Temp. Recorder Should be capable of printing Temp. Data in 0.1 0C increment. The thermocouples to be calibrated are placed in a highly stable temp. source (Controlled Ice point device, not reference device or controlled temperature bath) along with the reference standard. The Diff. in reading B/w the TC & reference device are recorded calibration of TCs should be carried out at tow temp before & after validation (1) at ice point at 0c(2) hot point slightly highest than the expected sterilization temp 1251300C..Calibration should be repeated after a series of validation TCs are calibrated once a week.

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Regulatory Requirement of Sterilization Process Validation

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10. Heat Distribution Study(2,3,9,11,15)

This study has traditionally been considered a critical aspect of sterilizer qualification. The intent of this study is to demonstrate the temperature uniformly & stability of the sterilizing medium throughout the sterilized. Heat distribution study include tow phases 1. 2. Heat distribution in an empty autoclave chamber. heat distribution in a loaded autoclave chamber.

Between 10-20 thermocouples should be used per cycle. Thermocouples should be secured inside the chamber according to definite arrangement. Teflon taps can be used to secure thermocouples. The trips where wires are soldered should not make contact with the autoclave inferior walls or any metal surface. One thermocouple each should remain in an ice bath & high temp oil bath during each cycle for reference when the temp. Monitoring equipment has the capability for electronically compensating each temp measurement against a internal surface. In initial study empty fewer thermocouple as the cold spot in the chamber & in the load is identify. Purpose of this to identity on a reproducible basis the location of the cool spot & effect of the load size/ configuration on the cool spot location. Minimum & maximum load size in the proper configuration elucidating where the cool spot is located. Three consecutive successful runs are performed for each cycle type with typical acceptance criteria. Difference in temp B/w the coolest spot & the mean chamber temp Should be not greater than +2.5OC .All temp measure in the chamber do not fluctuate by more than 1OC. The interval of time b/w the attainment of sterilization temp in the hottest & coldest part of chamber does not except 15 Sec. for chambers of to more than 800 L & not to exceed 30 Sec. for larger chamber. Time measurement shall be controlled to an accuracy of + 1 %.

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Regulatory Requirement of Sterilization Process Validation

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The thermocouple used in heat distribution studies are distributed geometrically in representative horizontal & vertical planes throughout the sterilizer. An additional to should be placed in the exhaust drain.

Fig 5:- Suggested location the thermocouples on a single shell for heat distribution study in heat sterilizer.

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Regulatory Requirement of Sterilization Process Validation

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11.Heat Penetration Study(2,3,9)

This is the most critical component of the entire validation process. In this test FO value of the cold spot inside the commodity located at the cool spot which is determined by Heatdistribution studies. The Container > 100 ml cold spot is determined by container-mapping studies. Thermocouples probe are inserted with in a container & repeat cycle are run to establish the point inside the container that is coldest most of time. FO Value will be calculated based on the temperature recorded by the thermocouples inside the container at the collets area of the load. FO will indicate wheatear the cycle is adequate or inadequate. Loaded chamber steam penetration conducted on every load. It is used to determined which load item most difficult to sterilize & which location within the item presents the worst case condition Determination of most difficult load item & the location in load item evaluated on a case by case basis. Intent of Heat penetration studies is to conform that the slowest to heat object within a specified load has achieved the requisite lethality. Lethal rate determined form the temp. data obtained from the heat penetration study

T0 TB

= =

Temp. Within the object the object or container. Process temp ( 1210 C for steam sterilization)

Total Lethality of Cycle then F0 = E 10 (T-121)/10

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Regulatory Requirement of Sterilization Process Validation

t = Time Interval.

2009

Heat penetration study can only conform temperature and hot other condition required for effective moist heat sterilization.

12 Microbiological Challenge Test(2,3,8,9)

Heat penetration study only conform the temp for adequate lethality. So other condition required for effective moist heal sterilization microbial challenge test are employed to proved additional necessary assurance that adequate lethality for all part of load. Mostly microbial challenge test conducted parallel with heat penetration study. Calibrated Biological indicator used for this purpose function as bioburden modules providing data for calculation of FO Mostly high heat resistance microorganism used for challenge test like G. Stearothermophilus & clostridium progenies. Microbial challenge test provide the assurance of sterility of loading material. Microbiological challenge test are conducted after the determination of worst case item & worst case location. A Thermocouple should be placed along with each indicator, as the temp data required to extrapolate the cycle to achieve the SAL of 10-6.Test are conducted until a cycle time result in three consecutive runs where the biological indicator not show growth. Three consecutive successful biological challenge runs are performed for each load with typical acceptance criteria consist with the empty chamber distribution test acceptance criteria & all biological indicators used during test cycle must show negative growth. When including solid marital the spores can be introduced on the surface of the item. After the sterilization cycle is complete the inoculated item of spore strips are recovered & subjected to microbiological test procedure. Strips are immersed in a suitable growth medium ( Soybean Case in digest medium) & incubated for up to seven days. incubation temp for G. Stearothermophiles is 50-55OC. For over kill cycle it is expected that all spore strips will be negative. Both positive ( unutilized strips) & negative ( growth medium with no spores also incubate with challenge sample.
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Regulatory Requirement of Sterilization Process Validation

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13 Bowie Dick Test(4,11,13)

Rapid & even penetration of steam in to the all part of sterilization load is important factor for achieving & holding the sterilization time. Bowie dick test helps the sure to know whether or not steam penetration of test pack is even a rapid. presence of air pockets & non condensable gases in the load can be ensured by Bowie dick test. Principle:Test is based on the use of a chemical indicator in the form of an adhesive tape such to a piece of suitable paper to form a st. Andrew's cross. This indicator paper is placed at the center of the test pack of folded huckaback. The indicator tape shows a change of color is response to a combination of time, temperature & moisture, when no air present in chamber steam will penetrate rapidly & completely & the indicator will show a uniform color change. place the test pack in the chamber with the bottom of the pack supported 100-200 mm above the center of the chamber base holding time at 121OC is 17 min. maximum & 16.8 min. minimum.

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Regulatory Requirement of Sterilization Process Validation

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14 Definitions(9,12)
SAL : sterility assurance level. SAL of 10-6: the probability of a single viable microorganism being present is one in one million. Bioburden: the number/type of viable microorganism contaminating an item. Overkill Approach: a sterilization approach based on assuming worst-case conditions (a bioburden of 106 of a highly heat resistant bacteria) Log Reduction: Reduce the surviving microbial population by 1 log or decrease the surviving population by a factor of. 12-Log Reduction: the log reduction required achieving overkill and a SAL of 10-6. CFU : colony-forming unit. D-value: time in minutes, at a specific temperature, to reduce the surviving microbial population by 90% (one logarithmic reduction). Z-value: temperature change required resulting in a 1-log reduction in D-value. F-value: the number of minutes to kill a specified number of microorganisms with a specified Z-value at a specific temperature. F0-value: the number of minutes to kill a specified number of microorganisms with a specified Z-value of 10C(50F) at a temperature of 121.1C(250F). 1 F0: the equivalent of 1 minute at 121.1C (250F). Dwell period: the time period that begins when the autoclave temperature has reached the set-point and ends when the timer has expired.
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Regulatory Requirement of Sterilization Process Validation

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Worst case items: items in the load which are the most difficult to sterilize (as determined by steam penetration studies). Worst case location: the location within an item that is the most difficult to sterilize (as determined by steam penetration studies). Gravity Displacement: a method of removing air by introducing steam into the top of a chamber and displacing the air, which is heavier than steam, by removing the air from the bottom of the chamber. Vacuum Cycle: A sterilization cycle that draw one or more vacuums to remove air prior to starting the dwell period. Pre-vacuum : A vacuum drawn prior to starting the dwell period to remove it. Hard Goods cycle : A sterilization cycle designed for times for which air removal is not difficult and therefore generally one pre vacuum is drawn. Wrapped Goods Cycle : A sterilization cycle designed for items for which air removed is difficult and therefore generally three or more pre vacuums are drawn. Liquids Cycle : A cycle designed for liquid loads that generally uses gravity displacement rather than drawing a vacuum. Bowie Dick Test : A test designed to verify that an autoclaves vacuum phase is removing a sufficient amount of air prior to the introduction of steam into teh chamber and tests for air leaks into the chamber. Empty Chamber Tests : Tests with an empty chamber essentially designed to demonstrate that an autoclave provides a uniform sterilizing environment. Steam Penetration Test : Loaded Chamber tests designed to determine the worst case items and worst case locations within a load. Biological Challenge Tests : Loaded chamber test designed to challenge the worst case location (within worst case items with biological indicators to demonstrate the effectiveness of a sterilization cycle. Steam Integrators : commercially available indicators that provide an indication of exposure to steam.
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Regulatory Requirement of Sterilization Process Validation

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Fixed Load : A load configuration where the quantity and location of items within the chamber are fixed. SIP : Steam in place or sterilize in place (often used interchangeably although the level of microbial destruction achieved may differ.

15 LIST OF FIGURES
FIGURE Fig.1 Fig.2 Fig.3 Fig.4 Fig.5 PAGE NO. 13 22 24 29 34

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Regulatory Requirement of Sterilization Process Validation

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16 LIST OF TABLES

TABLE Table no.1 Table no.2 Table no.3 Table no.4 Table no.5

PAGE NO 13 16 17 19 19

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Regulatory Requirement of Sterilization Process Validation

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REFERENCES
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Regulatory Requirement of Sterilization Process Validation

12.

2009

Garfinkle B.D, Henley M.W., Sterilization, Genaro A. R., Remington : The Science & Practice & Pharmacy, Lippincott Williams & Wilkins, Vol-I, 20th edition 2000;753-60.

13.

Carter S.J., Cooper & Gun,s Dispensing for Pharmaceutical Students, CBS publishers & Distributors, New Delhi,1999; Twelfth edition;431.

14.

Denyer S.P., Hodges N.A. & Gorman.S.P., Hugo & Russels Pharmaceutical Microbiology, Blackwell publishing, 7th edition, 2004; 346-55

15.

General

guideline

for

manufacturing

validation,

Available

from

URL:http://www.Prismpharmatech.com [Accessed on 26 Aug, & 13 Sep 2009]. 16. Autoclave Validation Protocol, Available from

URL:http://www.gmponblog.vinvarun.biz. [Accessed on 16 Aug. &20 Sep. 2009] 17. Kumser S., Validation of Sterilization Equipments, Istanbul Hilton, May 2-3 2002;

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