Navana Pharmaceuticals Ltd.

Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

VALIDATION PROTOCOL
OF
STERILITY TEST BY MEMBRANE FILTRATION METHOD

Prepared By: _______________________

Md. Golam Faruque
Executive, Microbiology

Checked By: _______________________

Utpal Banik
Assistant Manager, Microbiology

Reviewed By: _______________________

Md. Mostfizur Rahman
Manager,QC

Reviewed By: _______________________

Shahana Shilpi
AGM, QC

Approved By: _______________________

M.Golam Sorwar Chowdhury
Head of QA
 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date

1. NTRODUCTION
The sterility test procedure specified for a product must be capable of detecting low number of viable
microorganisms that may be present in the specimen sample. This validation protocol details the procedure used in
the validation of the sterility test of the sterile products by direct inoculation method. Validation is to be performed
when the test for sterility has to be carried out on reformulated or new product, or whenever there is a change in
the experimental conditions of the test.
2. AIM
The objective of this validation protocol is to demonstrate that:
2.1 The described testing process removes or inactivates any antimicrobial activity of the product and also in the
product-test medium mixtures.
2.2 Product-test medium mixtures are capable of supporting the proliferation and subsequent detection of the
challenge organisms within in the short incubation period.
The sterility test procedure is deemed to be valid if there is sufficient proliferation to permit visual detection of all
the challenge microorganisms in the respective combination of test medium, product, inoculums and incubation
temperature within the incubation period.

3. PRINCIPLE
Before tests for sterility for any product are initially carried out, it is necessary to demonstrate the validity of the
test method used by recovery of a small number of micro-organisms in the presence of the product It is preferable
to add these challenge organisms directly to the product prior to membrane filtration; where this is not practicable
due to inhibition or irreversible binding by the product, the challenge organisms should be added to the last rinse
solution. Validation should mimic the test proper in every detail, such as in the volumes of media used, quantities
and dilutions of product and diluents: the approach depends on the method of test and details are given in each
section. It may be performed concurrently with the actual test for sterility but should be confirmed as successful
before the results of the sterility test are interpreted.

4. REFERENCE
4.1 USP 41, Chapter (71).
4.2 Pharmaceutical Microbiology Manual by Food and Drug Administration office of Regulatory Affairs , ORA.007 ,
25 August 2020

5. EQUIPMENT & APPARATUS

Laminar flow cabinet, Sterile Magnetic Filter Funnel, Sterile forceps and scissors, Sterile Gloves (Latex), Sterile
Absorbent Cotton.

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
6. FILTER PAPER
Use Individually packed sterile membrane filter having a nominal pore size not greater than 0.45 µm whose
effectiveness to retain micro-organisms has been established. Cellulose nitrate filter, for example, are used for
aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for example, strongly alcoholic
solutions. Specially adapted filters may be needed for certain products, e.g. for antibiotics.
Use hydrophobic edged membranes, Where the product has antimicrobial activity, so as to facilitate washing
and inactivate antimicrobial residue on filter paper.

7. PREPARATION OF CULTURE MEDIA

7.1 Culture medium for the growth of aerobic and anaerobic bacteria (Fluid Thioglycollate Medium, FTM): Mix
prescribed quantity of dehydrated powder of culture media as recommended by the manufacturer in purified
water, boil to dissolve the contents, cool & adjust pH with 1.0N NaOH to 7.1 ± 0.2 if required. Dispense 100mL or
required quntity of medium in screw capped container and sterilize at 121˚C for 15 minutes or validated cycle.
7.2 Medium for the growth of bacteria and fungi (Soybean-Casein Digest Medium, TSB)Mix prescribed quantity of
dehydrated powder of culture media as recommended by the manufacturer in purified water, boil to dissolve the
contents, cool & adjust pH with 1.0N NaOH to 7.3 ± 0.2 if required. Dispense 100mL or required quantity of
medium in screw capped container and sterilize at 121˚C for 15 minutes or any validated cycle.
7.3 If any neutralizing agent is added to the culture media, record it into the validation report.

8. PREPARATION OF DILULTING AND RINSING FLUID

8.1 Fluid A: Dissolve 1g of peptic digest of animal tissue in water to make 1 Liter, filter or centrifuge to clarify, if
necessary, and adjust to a pH of 7.1 ± 0.2 . Dispense into containers and sterilize using a validated process or at
121˚C for 15 minutes. For penicillins or cephalosporins aseptically add to the above preparation, if necessary, a
quantity of sterile β-lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the
solution has been filtered.
8.2 Fluid D: To each liter of Fluid A add 1 ml of polysorbate 80, adjust to a pH of 7.1 ± 0.2, dispense into containers
and sterilize using a validated process or at 121˚C for 15 minutes.
8.3 Fluid K: Dissolve 5 g of peptic digest of animal tissue, 3 g of beef extract and 10g of polysorbate 80 in water to
make 1 liter. dispense into containers and sterilize using a validated process or at 121˚C for 15 minutes.
8.4 Record the volume of diluents used into the validation report.

9. STERILISATION OF APPARATUS
Sterilize Forceps, Scissors, Absorbent Cotton, sterile Gloves, Sterile Duster at 21˚C for 15 minutes or any validated
cycle.

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
10. STERILISATION OF MEDIA
Prepare the media according the manufacturer instruction and sterilize the media by autoclaving at 121 degree
centigrade for 15 minutes or any validated cycle.

11. PREPARATION OF CHALLANGEING INOCULUM (10-100cfu)

11.1 Material Required: Tryptone soya Agar plates (TSA), sterile phosphate buffer solution, Sterile Petri Plates,
Measuring Cylinder.
11.2 Test Organisms: Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa ATCC 9027 Bacillus subtilis ATCC
6633 Clostridium sporogenes ATCC 19404 Candida albicans ATCC 10231 Aspergillus niger ATCC 16404.
11.3 Preparation of culture suspension:
Follow the below steps or follow the manufacturer instruction to obtain low inoculums such as 10 to 100 CFU
microorganisms
11.3.1. Remove the desired lyophilized culture container from the refrigerator and take the culture vial as well as
desired quantity of hydration fluid vial.
11.3.2. Allow the taken lyophilized culture to equilibrate at room temperature
11.3.3. Prepare the inoculum of 10-100 cfu/ ml according to the manufacturer instruction
11.3.4. Replace the rubber stoppers, recap the vial and return the remaining lyophilized culture in its original
container into the refrigerator.
11.3.5. This is the working culture suspension and this suspension contains 10-100 cfu/ml.
11.3.6. Use this suspension within 30 minutes to ensure microorganisms viability.
11.3.7. Transfer 1 ml from the working culture suspension and add to 100ml of sterile rinsing solution.
11.3.8. Prepare the inoculum of 10-100 cfu in duplicate, use one inoculum for proving that the inoculums was
challenged contained 10-100 cfu and use another inoculum for rinsing solution.
11.3.9. Confirm that this 100ml of rinsing solution contain 10-100 cfu by filtering the solution through 0.20 µm
membrane filter and place it on to the surface of TSA agar plates and incubate the plate according to the
following table:

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date

Table 1: Organisms and their incubation temperature & period

Organism Incubation Incubation Period
temperature
Staphylococcus aureus ATCC 30-35° C Not more than 3
6538 days
Clostridium sporogenes ATCC 30-35° C Not more than 3
19404 days
Pseudomonas aeruginosa ATCC 30-35° C Not more than 3
9027 days
Bacillus subtilis ATCC 6633 30-35° C Not more than 3
days
Candida albicans ATCC 10231 20-25° C Not more than 5
days
Aspergillus niger ATCC 16404 20-25° C Not more than 5
days

12. NUMBER OF ARTICLES TO BE TESTED

Table 2: Minimum quantity to be used for each Medium

Quantity per container Minimum quantity to be used

LIquid
Less than 1 mL The whole content
1-40 mL Half the contents of each container, but not
less than 1 mL
Greater than 40 mL, and not 20 mL
greater than 100 mL
Greater than 100 mL 10% of the container, but not less than 20 mL
Antibiotic liquids 1 mL
Insoluble preparations, ointments, Use the content of each container to provide
creams, to be suspended on not less than 200 mg
emulsion
Solids
Less than 50 mg Whole content of each container
50 mg or more, less than 300 mg Half the content of each container, but not less
than 50mg

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
300mg-5 g 150 mg
Greater than 5 g 500 mg

Table 3: Minimum number of Articles to be Tested in Relation to the Number of Articles in the Batch

Number of Items in the Batch* Minimum Number of Items to be

tested for Each medium
Parenteral preparation
Not more than 100 containers 10% or 4 containers, whichever is the
greater
More than 100 but not more than 500 10 containers
containers
More than 500 containers 2% or 20 containers, whichever is less
For large-volume parenterals 2% or 10 containers, whichever is less
Antibiotic solids
Pharmacy Bulk packages(<5 g) 20 containers
Pharmacy Bulk packages(>5 g) 6 containers
Bulk and blends See bulk solid products

Ophthalmic and other noninjectable preparation

Not more than 200 containers 5% or 2 containers, whichever is the
greater
More than 200 containers 10 containers
If the product is presented in the form of
single dose containers, apply the scheme
shown above for preparation for parenteral
use
Catgut and other surgical sutures for 2% or 4 Packages, whichever is the
veterinary use greater
Not more than 100 articles 10% or 4 articles, whichever is the
greater
More than 100 but not more than 500 10 articles
containers
More than 500 containers 2% or 20 articles, whichever is the less
Bulk solid products
Up to 4 containers Each container

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
More than 4 but not more than 50 20% or 4 containers, whichever is the
containers greater
More than 50 containers 2% or 10 containers, whichever is the
greater

13. PREPARATION OF STERILITY TEST SAMPLE

13.1 Aqueous solution: If appropriate, transfer a small quantity of a suitable, sterile diluents such as Fluid A, onto the
membrane in the apparatus and filter. The diluent may contain suitable neutralizing substances or appropriate
inactivating substances, for example, in the case of antibiotics. Transfer the contents of container or containers
to be tested to the membrane or membranes, if necessary, after diluting to the volume required, but using not
less than the quantities of the product examined prescribed in table-2 and table-3. Filter immediately, if the
product has antimicrobial properties, wash the membrane not less than three times by filtering through it each
time the volume of the chosen sterile diluent. Do not exceed a washing cycle of five times 100 ml per filter, even
if it has been demonstrated that such a cycle does not fully eliminate the antimicrobial activity.
13.2 Soluble solids : Use for each medium not less than the quantity prescribed in Table -2 and table- 3 of the
product dissolved in a suitable solvent such as a 1 g/l neutral solution of meat or casein peptone and proceed
with the test as described above for aqueous solutions using a membrane appropriate to the chosen solvent
13.3 Oils and oily solutions: Use for each medium not less than the quantity of the product prescribed in Table -2.
and table- 3. Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry
membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate
shown not to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the membrane by
its own weight then filter, applying the pressure or suction gradually. Wash the membrane at least 3 times by
filtering through it each time about 100 ml of a suitable sterile solution such as 1 g/l neutral meat or casein
peptone containing a suitable emulsifying agent at a concentration shown to be appropriate in the validation of
the test, for example polysorbate 80 at a concentration of 10 g/l. Transfer the membrane or membranes to the
culture medium or media or vice versa as described above
13.4 Ointments and creams: Use for each medium not less than the quantities of the product prescribed in Table 2.
Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent in isopropyl
myristate, by heating, if necessary, to not more than 40 °C. In exceptional cases it may be necessary to heat to
not more than 44 °C. Filter as rapidly as possible and proceed as described above for oils and oily solutions.
13.5 Solids for injection other tan antibiotics : constitute the test articles as directed on the label and proceed as
directed for aqueous solutions or oils and oily solutions, whichever applies.
13.6 Antibiotic solids for injection:
13.6.1 Pharmacy bulk packages,<5 g- from each 20 containers, aseptically transfer about 300 mg of solids, into
a sterile 200 ml of Fluid A and dissolve. Proceed as directed for Aqueous solutions or oils an oily
solutions, whichever applies.

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
13.6.2 Pharmacy bulk packages, ≥ 5g- from each of 6 containers, aseptically transfer about 1g of solids into a
sterile 200 ml of Fluid A and dissolve. Proceed as directed for Aqueous solutions or oils an oily solutions,
whichever applies.
13.6.3 Antibiotic solids, bulks and blends- Aseptically remove a sufficient quantity of solids from the
appropriate amount of containers, mix to obtain a composite, equivalent to about 6 g of solids, and
transfer to sterile 200 ml of Fluid A and mix. Proceed as directed for Aqueous solutions.

14. GROWTH PROMOTION TEST OF THE MEDIA (positive control without product)
14.1 Inoculate 100 ml of Fluid Thioglycollate Medium of the respective lot without product with inoculums of not
more than 100 cfu of Staphylococcus aureus ATCC 6538,Pseudomonas aeruginosa ATCC 9027, and Clostridium
sporogenes ATCC 19404 using separate portion of medium for each organism.
14.2 Inoculate 100 ml of Soyabean-Casein Digest Medium of the respective lot without product with prepared 10 to
100 cfu of Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 using
separate portion of medium for each organism
13.1 Incubate bacteria at 30-35°C for not more than 3 days and fungi at 20-25°C for not more than 5 days.
14.3 Check the incubated media after required days of incubation and record the results into the report.

15. NEGATIVE CONTROL ( Control Vessel without product)

Filter approximately 200 ml of sterile diluents chosen for diluting and washing of the respective product through
another separate filter unit autoclaved in the same cycle. Aseptically remove the membrane filter from the holder
and cut it into two pieces with sterile scissors. Immerse each portion of the membrane filter separately in sterile
100 ml of Fluid Thioglycollate Medium and Soya-bean Casein Digest Medium containers. Incubate Fluid
Thioglycollate media at 32±2.5˚C for 3 days and Soya-bean Casein Digest Medium at 22±2.5˚C for 5 days.

16. VALIDATION TEST

16.1 Sanitize the exterior part of the each sample with 70% Isopropyl Alcohol and place on the bench of Laminar Air
Flow (LAF).
16.2 Transfer the required quantity of sample as per Table 2 in 500ml screw cap conical flask containing sterile
respective diluents under LAF. Dissolve properly by gentle mixing.
16.3 Transfer the solution into sterile filtration unit containing sterile filter paper. Filter the whole content properly by
vacuum filtration unit. Wash the membrane filter with sterile respective rinsing solution.
16.4 Finally rinse the membrane filter by filtering through 100ml of respective solution containing 10 to 100 cfu one of
the challenge organism.
16.5 Record the total quantity is used to dilute or rinse the filter paper including the quantity of rinsing solution
containing the challenge organism into the validation report.
16.6 Repeat the same procedure for rest of the challenging organisms.
16.7 Aseptically remove the membrane filter from the holder and transfer the filter paper into the TSB in case of

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
Candida albicans ATCC 10231 , Aspergillus niger ATCC 16404 and Bacillus subtilis ATCC 6633.
16.8 Aseptically remove the membrane filter from the holder and transfer the filter paper into the FTM in case of
Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa ATCC 9027 and Clostridium sporogenes ATCC 19404.
16.9 Incubate Fluid Thioglycollate media at 32±2.5˚C for 3 days and Soya-bean Casein Digest Medium at 22±2.5˚C for
5 days in the case of fungus and at 32±2.5˚C for 3 days in the case of bacteria.

17. ACCEPTANCE CRITERIA FOR THE VALIDATION TEST

17.1 If clearly visible growth of micro-organism is obtained in the presence of the product after incubation, visually
comparable to that in the control vessel without product, either the product possesses no antimicrobial activity
or the activity has been satisfactorily eliminated. The test for sterility may then be carried out without further
modification.
17.2 If clearly visible growth of micro-organism is not obtained in the presence of the product to be tested, visually
comparable to that in the control vessel without product, the product possesses antimicrobial activity that has
not been satisfactorily eliminated under the condition of the test. Modify the conditions by using increasing the
number and volume of rinse solution in order to eliminate the antimicrobial activity and repeat the method
validation test. Do not exceed a washing cycle of 5 times 100 ml per filter, even if it has been demonstrated that
such a cycle does not fully eliminate the antimicrobial activity.
17.3 In case of growth promotion test, the media are suitable if clearly visible growth of the microorganism occurs
within the incubation period.
17.4 No growth should be found in the negative controls.

18. RE-VALIDATION CRITERIA

Re-validation is to be performed when the test for sterility has to be carried out on reformulated or new product,
or whenever there is a change in the experimental conditions of the test.

19. LIST OF DISTRIBUTION

Sl. Division/Department/Section Copy Sl. No. Division/Department/Section Copy No.

No. No.
01 QUALITY ASSURANCE N/A 03 QUALITY CONTROL CENTRAL 02
(Master Copy) LABORATORY (MICROBIOLOGY)
02 QUALITY CONTROL 01
CEPHALOSPORIN LABORATORY
(MICROBIOLOGY)

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 Navana Pharmaceuticals Ltd.
Method Validation Protocol Rupshi,Rupganj. Narayangonj, Bangladesh

Document No: AMVP/NP/MB(CEP)/Sterility_Filtration/077 Revision No 02

Issue Date 14 AUG 2022 Effective Date

Date
20. CHANGE HISTORY

Sl. No. Name of initiator Revision No. Effective date Reason for change
01 Utpal Banik 00 17 APR 2019 New
Md. Golam Faruque
02 01 Logo change and Routine Review.

21. ANNEXURE
Annex 1: Validation Report of Sterility Test by membrane filtration method

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