27/7/2017 SterlityValidation(MembraneFiltrationMethod)inPharmaceuticals:PharmaceuticalGuidelines

SterlityValidation(MembraneFiltrationMethod)inPharmaceuticals
Learn how to validate the method of sterility testing by membrane filtration method.
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 recovery of a few number 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 consisten
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 Equipments to be used for Sterility test validation are qualified and operational SOPs established and followed.
3. All the equipments 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 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 pe
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. Product Batch Batch Date of Bottles Sampled Sign

No. Name No. Size sampling sampled by
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 fina
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 35C orin SCDM and incubate at 20 to 25C as per table 1.
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 ste
and add an inoculum of viable cells of the specific bacteria and fungi respectively to FTM and SCDM and incubate at 30 to 35C and 20 to 25C respectivel
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 bacteri
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
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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 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
representative tube containing FTM that has been incubated at 3035C, an inoculum of viable cells of specific bacteria. To the next tube containing SCDM
that has been incubated at 2025C, 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 fo
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 Negative Product Positive Control Negative Control
Product Control Control
1 Rinse the Rinse the Take four tubes Rinse the
membrane with membrane with of FTM & three membrane with
approx 15 ml of approx 15 ml of tubes of SCDM. approx 15 ml of
sterile peptone sterile peptone sterile peptone
water water water
2 Select 20 bottles Select 20 bottles Add to each tube Rinse the
randomly and randomly and pull 10100 cfu of the membrane with
pull the half the half content full cultures listed in 100 ml sterile
content full content of table 1 peptone water
content of container in case of
container in case SVP into a filter
of SVP into a holder & start the
filter holder & filtration
start the
filtration.
3 Rinse the Rinse the Incubate the Aseptically cut the
membrane with membrane with 2 X SCDM tubes at filter paper into
2 X 100 ml 100 ml peptone 2025C for NMT two halves using
peptone water. water 5 days and FTM sterile S.S. Scissor
tubes at 3035C and transfer one
for NMT 3 days. half in sterile FTM
and one half in
sterile SCDM
media
4 Rinse the Finally rinse the Incubate the
membrane with membrane with 100 NA SCDM tubes at
100 ml of ml of sterile 2025C and FTM
peptone water, peptone water. tubes at 3035C
which is for 14 days
previously
inoculated with
10100 cells of
any one positive
cultures as listed
in table1.
5 Inoculate the Aseptically cut the
whole filter paper into two NA NA
membrane to halves using sterile
respective media S.S. Scissor and
tube and label transfer one half in
properly. Repeat sterile FTM and one
the same half in sterile SCDM
procedure for media.
remaining
microbial strains
as listed in
Table1.
6 Incubate the Incubate the SCDM
SCDM tubes at tubes at 20 25C NA NA
20 25C for and FTM tubes at
NMT 5 days and 3035C for 14
FTM tubes at 30 days.
35C for NMT 3
days.
Table I
Sr. No Positive control Fluid Thioglycollate Soyabean Casein Digest
Medium Medium

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1 Positive Control S. aureusNCIM 2079 C. albicansNCIM 3471
I
2 Positive Control Ps. aeruginosaNCIM A.nigerNCIM 1196
II 2200
3 Positive Control B. subtilisNCIM 2063 Environmental Flora EF II Acceptance Criteria
III 1. If the conspicuous growth is observed within 3 days for
4 Positive Control Environmental Flora EF bacteria and 5 days for fungi, and the growth of each
IV I 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 o
the test or such a 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 Product Control Positive Control Negative Control
No
1 Rinse the membrane with approx NA Negative Control I
15 ml of sterile peptone water Rinse the membrane with
approx 15 ml of sterile
peptone water
2 Select 20 bottles randomly and NA Rinse the membrane with
pull the half content full content 100 ml sterile peptone
of container in case of SVP into a water
filter holder & start the filtration
3 Rinse the membrane with 3 X 100 NA Aseptically cut the filter
ml peptone water 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 NA Incubate the SCDM tubes
two halves using sterile S.S. Scissor at 20 25C and FTM tubes
and transfer one half in sterile FTM at 3035C for 14 days
and one half in sterile SCDM
5 Repeat the same procedure for NA NA
remaining microbial strains as
listed in Table1.
6 Incubate the SCDM tubes at 20 NA NA
25C and FTM tubes at 3035C
for 14 days
7 After incubation observe the tubes NA NA
for growth. Growth Should be
absent
8 After 14 days incubation, Take four tubes of FTM & Negative Control II
separately add an inoculum of three tubes of SCDM. Take one one tube of FTM
viable cells of microorganisms 10 & SCDM.
100 cells to the FTM and SCDM
tubes, listed in Table 1.
9 Return all the inoculated tubes to Add to each tube 10100 Add to each tube 1010 ml
their previous temperature and cfu of the cultures listed in sterile WFI.
continue incubation 3 days for table 1
FTM and 5 days for SCDM.
10 NA Incubate the SCDM tubes Incubate the SCDM tube at
at 20 25C for NMT 5 days 20 25C for 5 days and
and FTM tubes at 3035C FTM tubes at 3035C for 3
for NMT 3 days. days.
Acceptance Criteria:
1. 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:
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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 full incubation
period. It is also necessary to demonstrate that growthpromoting 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 andSoybeancaseindigest media are 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 Antimicrobia
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 absence of growth.
3. After confirmation of 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 mlnormal salinecontaining 5 test tubes, 45 mlnormal salinecontaining 1 test tube & 90 mlnormal salinecontaining 3 test tubes, plug
the tubeswith cotton plug and sterilize in an autoclave at 121C temperature for 20 minutes.
10.3 Sterilized Petri plates in hot air oven at 180C 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 ofnormal saline up to 104dilution, from 104dilution take 5 ml
and add it to 45 ml sterilenormal saline105 & from 105dilution take 10 ml and add it to 90 ml sterile normal saline 106 & repeat the procedure till
108dilution.
10.8 Preserve the original undiluted culture suspension & 105to 108dilutions in a refrigerator between 2C to 8C.
10.9 Aseptically pour 1 ml each of 103to 108dilution of each organism in each of the sterilized dry petridishes.
10.10 Aseptically pour in each plate about 20 ml sterilized Soybean Casein Digest agar medium for bacterial culture and sterilized Sabouraud Dextrose aga
for fungal culture, previously liquefied and cooled to around 45C, mix and allow the medium to solidify. Incubate the Plates at 32.52.5C for 24 to 48
hours for bacterial culture and 22.52.5C for 48 to 72 hours forfungal culture.
10.11 At the end of incubation count the number of Colony Forming Units developed on each of the petridishes.
10.12 Select the culture suspension containing 10100 cfu/ml and preserve in refrigerator between 2C to 8C.
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 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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