Sterility Validation (Membrane Filtration Method) in Pharmaceuticals

1.0 INTRODUCTION

When any test for sterility is initially carried out for any product, it is necessary to validate the test method used, by the recovery of a few numbers of microorganisms in the presence of the product.

2.0 OBJECTIVE

The Objective of this validation is to establish documented evidence that the test for sterility by membrane filtration method will produce the consistent results when analyzed as per the Standard Operating Procedure.

3.0 SCOPE

This protocol is applicable for Sterility test of the products.

4.0 RESPONSIBILITIES

S. No.	Responsibilities	Name of the Department
1	Preparation of Validation Protocol	Quality Control
2	Execution of Protocol	Quality Control
3	Approval of Protocol and Report	Quality Assurance
4	Final System Approval	Head QA / QC
5	Review and maintenance of Report	Quality Control

5.0 PREREQUISITES

In order to efficiently conduct validation of the Sterility Test by Filtration method, ensure that the following requirements are fulfilled.
1. Validated Aseptic facility to carry out the Sterility test Validation
2. All equipment to be used for Sterility test validation are qualified and operational SOP’s established and followed.
3. All the equipment and culture media required for the validation of sterility test should be sterile.
4. Trained personnel for conducting Sterility test and its Validation.
5. Membrane filter: Sterile individually packed cellulose nitrate or cellulose acetate, 47 mm diameter, average pore size 0.45m.
6. Sterilized filtration assembly, forceps and scissor
7. Sterile 70% IPA solution

6.0 EQUIPMENT / SYSTEM DESCRIPTION

1	Location	Sterility Room of Quality control department
2	Equipment	The equipment covers the Stainless steel filtration assembly with manifolds, Vacuum pump, Autoclave, Hot air Oven, Laminar air flow , and Incubator

7.0 IDENTIFICATION OF CRITICAL CONTROL / MONITORING PARAMETER

Before proceeding for sterility test Validation, following parameters to be checked
7.1 Each lot of dehydrated media used for the preparation of sterility test medium must be tested for its growth-promoting qualities as per SOP.
7.2 During validation carry out environmental monitoring by settle plate (Plate Exposure) and Personnel monitoring by finger dab and swab method as per SOP.
7.3 If any cfu observed during monitoring on LAF and Finger dab, all cfu must be identified up to species level.

8.0 SAMPLING DETAILS

1 Carry out the sampling of three consecutive batches from various sites throughout the sterilizer load.
2 Immediately carry out the leak testing and visually examine the bottles for any leakage or any extraneous particles.

S. No.	Product Name	Batch No.	Batch Size	Date of sampling	Bottles sampled	Sampled by	Sign
1.
2.
3.

9.0 VALIDATION TEST

Validation of Sterility Test by Membrane Filtration method is done by following procedures

A. Test for Residual Antimicrobial Activity

The Test for Residual Antimicrobial Activity is carried out the test procedure as described in general sterility test, up to the final wash procedure. To the final wash add an inoculum of viable cells of the specific bacteria and fungi. After the final wash with the added microorganisms has been passed through the filter, inoculate filter paper in FTM & incubate at 30 to 35ºC or in SCDM and incubate at 20 to 25ºC as per table 1.

The growth of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified.

B. Test for Antimicrobial Activity

To demonstrate that the mixture does not manifest antimicrobial activity, carry out the test as described in sterility test procedure, up to the incubation step and add an inoculum of viable cells of the specific bacteria and fungi respectively to FTM and SCDM and incubate at 30 to 35ºC and 20 to 25ºC respectively.

Growth of each of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified

C. Stasis Test (Efficacy of the test media at the end of incubation period)

The Stasis Test is designed to demonstrate that the media (i.e. FTM and SCDM) inoculated with the test preparations will support growth for the full incubation period. After incubation of the media has been completed in accordance with the instruction given in the sterility test for negative control, add to a representative tube containing FTM that has been incubated at 30-35ºC, an inoculum of viable cells of specific bacteria. To the next tube containing SCDM that has been incubated at 20-25ºC, add an inoculum of viable cells of specific fungi. Return all the inoculated tubes to their previous temperature and incubation continued.

All the tubes should show growth of added microorganisms within 48 hours. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test is considered invalid.

A. TEST FOR RESIDUAL ANTIMICROBIAL ACTIVITY)

Objectives:

The test is performed to ensure that; any residual of Antimicrobial Activity is satisfactory eliminated by using the steps mentioned in this protocol.

Procedure:

Step	Positive Product Control	† Negative Product Control	Positive Control	Negative Control
1	Rinse the membrane with approx 15 ml of sterile peptone water	Rinse the membrane with approx 15 ml of sterile peptone water	Take four tubes of FTM & three tubes of SCDM.	Rinse the membrane with approx 15 ml of sterile peptone water
2	Select 20 bottles randomly and pull the half content (full content of container in case of SVP) into a filter holder & start the filtration.	Select 20 bottles randomly and pull the half content (full content of container in case of SVP)  into a filter holder & start the filtration	Add to each tube 10-100 cfu of the cultures listed in table –1	Rinse the membrane with 100 ml sterile peptone water
3	Rinse the membrane with 2 X 100 ml peptone water.	Rinse the membrane with 2 X 100 ml peptone water	Incubate the SCDM tubes at 20-25ºC for NMT 5 days and FTM tubes at 30-35ºC for NMT 3 days.	Aseptically cut the filter paper into two halves using sterile S.S. Scissor and transfer one half in sterile FTM and one half in sterile SCDM media
4	Rinse the membrane with 100 ml of peptone water, which is previously inoculated with 10-100 cells of any one positive cultures as listed in table–1.	Finally, rinse the membrane with 100 ml of sterile peptone water.	NA	Incubate the SCDM tubes at 20-25ºC and FTM tubes at 30-35ºC for 14 days
5	Inoculate the whole membrane to respective media tube and label properly. Repeat the same procedure for remaining microbial strains as listed in Table-1.	Aseptically cut the filter paper into two halves using sterile S.S. Scissor and transfer one half in sterile FTM and one half in sterile SCDM media.	NA	NA
6	Incubate the SCDM tubes at 20- 25ºC for NMT 5 days and FTM tubes at 30-35ºC for NMT 3 days.	Incubate the SCDM tubes at 20- 25ºC and FTM tubes at 30-35ºC for 14 days.	NA	NA

 Table - I

Sr. No	Positive control	Fluid Thioglycollate Medium	Soyabean Casein Digest Medium
1	Positive Control – I	S. aureus NCIM 2079	C. albicans NCIM 3471
2	Positive Control – II	Ps. aeruginosa NCIM 2200	A. niger NCIM 1196
3	Positive Control – III	B. subtilis NCIM 2063	Environmental Flora– EF II
4	Positive Control – IV	Environmental Flora – EF I

Acceptance Criteria

1. If the conspicuous growth is observed within 3 days for bacteria and 5 days for fungi, and the growth of each challenge microorganisms in the Positive Product control containers are visually comparable to the growth in the positive control and there is no growth in negative control & negative product control, the product possess no antimicrobial activity under the condition of the test or such an activity has been satisfactory eliminated. The test for sterility may be carried out routinely without further modifications.

2. If the conspicuous growth is not observed within 3 days for bacteria and 5 days for fungi, or growths of each test organism in the Positive Product Control containers are visually not comparable with positive control containers respectively, the product possesses antimicrobial activity that has not been satisfactory eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the validation test. (i.e. by using additional washes)

Negative Product Control Test

The result of negative product control test facilitates the interpretation of sterility test results, particularly when used to declare a test invalid because of contamination in negative product control. The essential element of the negative control is to simulate the testing method.

B. TEST FOR ANTIMICROBIAL ACTIVITY

Objectives

The test is performed to ensure that, the absence of Antimicrobial Activity under the experimental conditions.

Procedure

Step No	Product Control	Positive Control	Negative Control
1	Rinse the membrane with approx 15 ml of sterile peptone water	NA	Negative Control I – Rinse the membrane with approx 15 ml of sterile peptone water
2	Select 20 bottles randomly and pull the half content (full content of container in case of SVP)  into a filter holder & start the filtration	NA	Rinse the membrane with 100 ml sterile peptone water
3	Rinse the membrane with 3 X 100 ml peptone water	NA	Aseptically cut the filter paper into two halves using sterile S.S. Scissor and transfer one half in sterile FTM and one half in sterile SCDM media
4	Aseptically cut the filter paper into two halves using sterile S.S. Scissor and transfer one half in sterile FTM and one half in sterile SCDM	NA	Incubate the SCDM tubes at 20- 25ºC and FTM tubes at 30-35ºC for 14 days
5	Repeat the same procedure for remaining microbial strains as listed in Table-1.	NA	NA
6	Incubate the SCDM tubes at 20- 25ºC and FTM tubes at 30-35ºC for 14 days	NA	NA
7	After incubation observe the tubes for growth. Growth Should be absent	NA	NA
8	After 14 days incubation, separately add an inoculum of viable cells of microorganisms (10 – 100 cells) to the FTM and SCDM tubes, listed in Table –1.	Take four tubes of FTM & three tubes of SCDM.	Negative Control II – Take one – one tube of FTM & SCDM.
9	Return all the inoculated tubes to their previous temperature and continue incubation 3 days for FTM and 5 days for SCDM.	Add to each tube 10-100 cfu of the cultures listed in table –1	Add to each tube 10-10 ml sterile WFI.
10	NA	Incubate the SCDM tubes at 20- 25ºC for NMT 5 days and FTM tubes at 30-35ºC for NMT 3 days.	Incubate the SCDM tube at 20- 25ºC for 5 days and FTM tubes at 30-35ºC for 3 days.

Acceptance Criteria

1. The growth of each of the added microorganisms in Product Control should be apparent within 3 days for bacteria and 5 days for fungi & should be comparable to positive controls.
2. If growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified (e.g. using additional washes) until growth does occur when tests as above are carried out.
3. Growth should be absent in Negative Controls.

C. STASIS TEST (Efficacy of the test Media at the end of Incubation Period)

Introduction

The Stasis Test is designed to demonstrate that the media (i.e. FTM and SCDM) inoculated with the test preparations will support growth for the full incubation period. It is also necessary to demonstrate that growth-promoting qualities of media are retained and stable for the full test period.

Objectives

The test is performed to ensure that, the growth-promoting qualities of fluid thioglycollate and Soybean casein digest media is stable for the full test period.

Procedure

1. Carry out the negative control for FTM (4 tubes) and SCDM (3 tubes) along with the Test for Residual of Antimicrobial Activity and Test for Antimicrobial activity without any inoculation up to the incubation Period.
2. After incubation, observe all the negative control of each medium i.e. FTM and SCDM for the absence of growth.
3. After confirmation of the absence of growth, add aseptically 10 to 100 viable cells of microorganisms in their respective tubes as described in Table – I.
4. Return the tubes to their previous temperature and continue the incubation for NMT 3 days for bacteria and NMT 5 days for fungi.
5. Observation: daily observe the tubes for evidence of microbial growth by means of turbidity.

Acceptance Criteria

1. The test is valid if the growth of each of the added microorganisms observed within 3 days for bacteria and 5 days for fungi.
2. If growth is not observed within 3 days for bacteria and 5 days for fungi the test is considered invalid.

10.0 METHOD FOR PREPARATION OF CELL SUSPENSION

10.1 Prepare soybean casein digest Agar medium & Sabouraud Dextrose agar medium in a conical flask as per Media Preparation SOP.
10.2 Prepare 0.9 % w/v solution of sodium chloride with distilled water in a conical flask & transfer to the test tubes in the following manner (for one microorganism): 9 ml normal saline containing 5 test tubes, 45 ml normal saline containing 1 test tube & 90 ml normal saline containing 3 test tubes, plug the tubes with cotton plug and sterilize in an autoclave at 121ºC temperature for 20 minutes.
10.3 Sterilized Petri plates in a hot air oven at 180ºC for 1 hour.
10.4 Perform the activity of serial dilution in the microbiological room.
10.5 Remove culture slant from the refrigerator i.e. MC1/M1/W1/D1, and allow it to attain ambient temperature.
10.6 Transfer 1 ml of sterile normal saline to the slant of freshly grown culture, mix using inoculation loop and suspend it in 9 ml sterile normal saline. Homogenize the suspension by gentle shaking.
10.7 Prepare serial dilutions (by diluting each time 1 ml of culture suspension with 9 ml of normal saline) up to 10-4 dilution, from 10-4 dilution take 5 ml and add it to 45 ml sterile normal saline (10-5) & from 10-5 dilution take 10 ml and add it to 90 ml sterile normal saline (10-6) & repeat the procedure till 10-8 dilution.

10.8 Preserve the original undiluted culture suspension & 10-5 to 10-8 dilutions in a refrigerator between 2ºC to 8ºC.
10.9 Aseptically pour 1 ml each of 10-3 to 10-8 dilution of each organism in each of the sterilized dry Petri dishes.
10.10 Aseptically pour in each plate about 20 ml sterilized Soybean Casein Digest agar medium for bacterial culture and sterilized Sabouraud Dextrose agar for fungal culture, previously liquefied and cooled to around 45ºC, mix and allow the medium to solidify. Incubate the Plates at 32.5±2.5ºC for 24 to 48 hours for bacterial culture and 22.5±2.5ºC for 48 to 72 hours for fungal culture.
10.11 At the end of incubation count the number of Colony Forming Units developed on each of the Petri dishes.
10.12 Select the culture suspension containing 10-100 cfu/ml and preserve in the refrigerator between 2ºC to 8ºC.
10.13 A volume of individual suspensions so prepared equivalent to about 100 cells of each organism per ml shall be used for Sterility Test Validation.

11.0 PRECAUTION

1. Validation tasks are to be carried out by trained personnel using techniques and equipment, which minimize the risk of accidental microbial contamination of the test and of the testing environment.
2. Validation tests should be conducted in Sterility Test Room and positive control tests in the microbiological testing room under LAF.
3. Personnel conducting sterility testing or associated aseptic manipulations should wear sterilized garments.
4. All equipment, vessels and materials, which are used for validation of sterility test, should be sterilized by autoclaving or by dry heat sterilization.

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