regulation of sterlization

Regulatory Requirement of Sterilization Process Validation 2009

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

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 Administration’s) 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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Analytical method [21 CFR 211 Sub Part I]

Equipment cleaning & maintenance [21 CFR 211.67]

Change control [21 CFR 211.100]

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. To built sterility in to product.

2. 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.

2. GUIDANCE FOR INDUSTRY FOR THE SUBMISSION OF DOCUMENTATION

FOR STERILIZATION VALIDATION 2


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

without the manufacture of three production batches. Sterilization process validation data,

however, should be generated using procedures and conditions that are fully representative

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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 21 CFR 312.23(a)(7)

New drug applications 21 CFR 314.50

Abbreviated new drug and abbreviated

antibiotic drug applications 21 CFR 314.94 and 314.50

Supplements to NDA's and ANDA's 21 CFR 314.70

2. Animal Drugs:

Investigational new animal drug applications 21 CFR Part 511

New animal drug applications 21 CFR 514.1

Supplements to NADA's 21 CFR 514.8

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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

A description of representative autoclave loading patterns should be provided.

5. Methods and Controls to Monitor Production Cycles

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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

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),

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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 bioburden-

based 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.

2. Specifications for Bioburden

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

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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, in-

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

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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

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

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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.

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.

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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 container-

closure system barrier over the claimed shelf life should be described.

B. Radiation

1. The Facility and the Process

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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 container-

closure system barrier over the claimed shelf life should be described.

3. AUTOCLAVE VALIDATION PROTOCOL (9,3,2,16)

Validation of the Autoclave is classified into the following

DQ- Design qualification

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IQ- Installation qualification

OQ – Operational Qualification

PQ – Performance Qualification

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: TABLE-1

NAME DEPARTMENT DESIGNATION ROLE &

RESPONSIBILITY

SIGN.

Q.C microbiologist Prep ration

Q.C Manager

microbiology

Protocol checking

(documentation of result)

Q.C manager Co-ordination & checking

maintenance manager Utility support

Q.C General manager approval

Q.A Sr. manager authorization

OPERATIONAL QUALIFICATION PROTOCOL (OQ)

PURPOSE :

To demonstrate and document that the operations of the Autoclave take place as specified .

SCOPE :

Autoclave xxxxx will be qualified to meet OQ.

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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 party’s 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 121-124°C and pressure must be within 15 to 18 lbs for

15min cycle.

12

8

9 11

6 5

10

7

4 1 3 2

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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 day’s 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.

Test name: TABLE-2 THERMOCOUPLE CALIBRATION

time Probe temp(oc) Display

temp(0c)

pressure

(psi/lb2)

Log time

1 2 3 4 5 6 7 8 9 10 11 12

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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.


All 12 probes must reach temperature 121°C to 124°C 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 – 25°C for 120 hrs


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

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temperature at 15psi and 121°C during the heat penetration studies, once on the maximum

load.


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 LOAD PATTERN CRITARIA

Heat

distributio

n

Empty load:test-1

All 12 probes must reach temp121oc to 124oc & pressure must be with in 15 -18 lbs for 15 min. cycle

Minimum load: test-1

Maximum load:test-1

Maximum load:test-2

Maximum load:test-3

Heat

penitration





Maximum load:test-1

Maximum load:test-2

Maximum load:test-3

Microbial

limit tests

Maximum /minimum load Negative control & nutritive

properties must pass

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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

Test name: TABLE-4 THERMOCOUPLE CALIBRATION

time Probe temp (oc) Display

temp(0c)

pressure

(psi/lb2)

Log time

1 2 3 4 5 6 7 8 9 10 11 12

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TABLE-5 TESTS INVOLVED IN AUTOCLAVE VALIDATION

TESTS LOAD PATTERN CRITARIA

Heat distribution Empty load:test-1



Maximum load:test-1

Maximum load:test-2

Maximum load:test-3

Heat penitration Minimum load: test-1




Maximum load:test-1

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Maximum load:test-2

Maximum load:test-3

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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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 :

D= Ulog N 0−log Nu

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 121°C the microbial population was reduced

2 × 105 to 6 × 103 then D value is :-

D= Ulog N 0−log N

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D121=5

log(2×105 )−log(6×103 )=3. 25 min

Thus at 121°C 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

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Spore dots

Spore suspension

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 .

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5. Z value (Resistance Value)(2,3,10)

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 10°C the accepted standard for steam sterilization of Bacillus sterothermophilus

spore & for a Z value of 20°C 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.

Z=T 2T 1

log D1−log D2 Equ…………… (2)

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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 :-

Steam sterilization Z = 10° C(18° F)

Dry Heat sterilization Z = 20°C (36° F)

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6. F Value / Lethality Value(2,3,4,10)

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. 121°C delivered to

container of unit of product.

. Equ…………. (3)

TO = 121°C for steam sterilization

TO = 170°C 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 121°C.

F value is used as a measurement of sterilization effectiveness FO is defined as the number

of equivalent minutes of steam sterilization at temp. 121.1°C delivered to a container or unit

of product calculating z value of 10°C.

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 121.1°C 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 20°C.

Mathematical FO

In mathematical terms, FO is expressed as :

FO=Δt×10(T−121. 1)/Z

for steam sterilization

FO=Δt×10(T−170)/Z

for Dry Heat sterilization

.t = time interval between measurements of T.

26


T = temp. of sterilized product at time t.

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 121.1°C

FO=Δt×10(T−121. 1)/Z

Z = 10 in steam sterilization

FO=12×10121.1−121.1/10

FO=12×10 °

FO=12 min

Exp 2 : t = 12 min at 120.1°C

FO=12×10(120. 1−121. 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.

FO=D121.1( log10A −log10

B ) Equ……….. (4)

D121.1 = D value at 121.1°C

A = Bioburden per container

B = Maximum acceptable sterility assurance level (SAL)

27


Exp : The product being sterilized has a bioburden of 100 spores per container, D value at

121.1°C is 3.3 min & Desire Acceptable sterilization limit is 10–6 So

FO=D121. 1° C ( log10A − log10

B )

FO=(3 . 3) [ log10100−log10

−6 ]

FO=(3 . 3) [2−[6 ] ]

FO=3 .3×8=2. 64 min

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 121°C.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. Container size, geometry & Heat transfer coefficient

2. Product volume & viscosity

3. Size and configuration of the batch load in sterilization

28


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 10–6.

It is desirable that B be as low possible. This may accomplished in one of two ways

29


(i) Reducing the Bioburden of bulk product.

(ii) Increase the equivalent exposure time.

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) Overkill cycle approach

(ii) BI/Bioburden cycle approach

(iii) Bioburden approach

A sterilization cycle means time required for sterilization of a product to produce a SAL of

10–6 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

30


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 10–6 d

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

31


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.

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 0.10C

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 125-

32


1300C..Calibration should be repeated after a series of validation TCs are calibrated once a

week.

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. Heat distribution in an empty autoclave chamber.

2. 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 33


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 %.

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.

34


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 Heat-

distribution 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

35


T0 = Temp. Within the object the object or container.

TB = Process temp ( 1210 C for steam sterilization)

Total Lethality of Cycle then

F0 = E 10 (T-121)/10

t = Time Interval.

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.

36


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.

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.

37


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.

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.

38


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 10°C(50°F) at a temperature of 121.1°C(250°F).

1 F0: the equivalent of 1 minute at 121.1°C (250°F).

Dwell period: the time period that begins when the autoclave temperature has reached the

set-point and ends when the timer has expired.

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.

39


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 autoclave’s 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.

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 PAGE NO.

Fig.1 13

Fig.2 22

Fig.3 24

Fig.4 29

Fig.5 34

40


16 LIST OF TABLES

TABLE PAGE NO

Table no.1 13

Table no.2 16

Table no.3 17

Table no.4 19

41


Table no.5 19

REFERENCES

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Processes, Informa Health Care USA, New York,2008;3rd edition,159-186

3. Nash R.A. & Wachter H.A., Pharmaceutical Process Validation, Marcel Dekker Inc.

New York;2003;83-110.

4. Sarant A.D., The Pharmaceutical Sciences Pharma Pathway, Pragati Books Pvt.

Ltd., Third Edition,2006;2.17-2.18

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5. Ference B. M., Kot L., Thomas R., Equipment Validation, Wiley,2003;351-68.

6. Hurber L.,Validation and Qualification in Analytical Laboratories, Informa Health

Care, New York,2002; Second edition ; 253.

7. Jayaram T., Good Validation Practices, Pharma Times, Vol-37, No.-9, Sept 2005,

13-16.

8. Alferdo J., Autoclave Validation, Available From URL: http: // www.Corp

Validation.com [Accessed on 18 Aug. & 20 Sep. 2009].

9. Lewis R.G., Practical Guideline to Autoclave Validation, Available From

URL:http:www.Ide-ch2m.com [Accessed on 18 Aug.&24 Sep.]

10. Akers M.J. & Avis K. E., Sterilization, Lackman L., Lieberman H. A., Kaing J. L.,

The Theory & Practice of Industrial Pharmacy, Vargesh Publishing House, Bombay;

, 1990;Third edition; 619-25.

11. Pelczar M.J., Chan E.C.S., Krieg N. R., Microbiology, Tata McGraw-Hill

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12. Garfinkle B.D, Henley M.W., Sterilization, Genaro A. R., Remington : The Science

& Practice & Pharmacy, Lippincott Williams & Wilkins, Vol-I, 20 th edition

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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 & Russel’s Pharmaceutical

Microbiology, Blackwell publishing, 7th edition, 2004; 346-55

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URL:http: // www.Prismpharmatech.com [Accessed on 26 Aug, & 13 Sep 2009].

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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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