A Sterility Test is a critical procedure in pharmaceutical and biopharmaceutical industries to ensure that large volume parenteral products are free from viable microorganisms. The filtration method is commonly employed for the sterility testing of large volume products. Below is a general outline for an SOP for Sterility Test of Large Volume Products by Filtration Method

PURPOSE

1. To assure the sterility of aseptically, terminal sterilized filled large volume products.
2. To establish a standardized procedure for conducting Sterility Tests on large volume parenteral products.
3. To ensure the absence of viable microorganisms in large volume products intended for parenteral administration.

SCOPE

1. This SOP is applicable to aseptically, terminal sterilized filled large volume products.
2. The procedure covers the preparation of test samples, filtration, and incubation of membranes for microbial examination.

RESPONSIBILITIES & AUTHORITIES

1. Designated persons from departments will prepare SOPs of their departments.
2. Departmental Managers will review and signed the SOPs.
3. Departmental Head will approve the SOPs.
4. Head of QA will authorize the SOPs.
5. Head of QA/Designee responsible to audit compliance.

REFERENCES & ATTACHED DOCUMENTS

1. Sterility Tests – USP <71>
2. Sterilization and sterility assurance of compendia method USP <1211>

DEFINITIONS & ABBREVIATIONS

Sterility Test:

Sterility is the test in which we ensure that the batch of product is sterile or has been sterilized”. The test is applied to substance, preparations or article which, according to the pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined in the condition of test.

PROCEDURE Sterility Test of Large Volume Products by Filtration Method

Requirements.

1. Sterile Millipore Filtration unit,
2. Sterile S.S Forceps,
3. Vacuum Pump,
4. S.S Manifold holder
5. Sterile glass syringes.
6. 0.45µ cellulose nitrate filter paper (sterile),
7. Sterile Glass media bottle.
8. Peptone water
9. Isopropyl Alcohol 70%
10. Fluid Thioglycollate Medium,
11. Soyabean Casein Digest Broth

Sterility of Media Sterility Test of Large Volume Products by Filtration Method

1. Confirm the sterility of media by incubating one container of each medium at the specified incubation temperature for not less than 14 days and by incubating un-inoculated containers as negative controls during sterility test procedure.
2. Fluid Thioglycollate Medium at 30 – 35°C.
3. Soyabean Casein Digest Broth at 20 – 25°C.

Diluting and Rinsing Fluid for Membrane Filtration:

Fluid A:

Dissolve 1g of Peptic digest of animal tissue in Purified water to make 1 liter (quantity to be adjusted as per requirement). Filter or centrifuge to clarify if necessary and adjust to pH 7.1 ± 0.2. Dispense into containers and sterilize by autoclaving for 15-20minutes at 121°C (15 lb/psi).

Fluid D:

To each liter of Fluid A, add 1ml of polysorbate 80(Amber colored liquid), adjust to a pH of 7.1 ± 0.2, dispense into containers, and sterilize using a validated process.

Fluid K Sterility Test of Large Volume Products by Filtration Method

Dissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 litre. Adjust the pH to obtain, after sterilization, a pH of 6.9 ± 0.2, dispense into containers, and sterilize using a validated process.

Method Suitability Test:

1. This method suitability is performed, when the test for sterility to be carried out on a new product.
2. Whenever there is a change in the experimental conditions of the test.
3. This method of suitability may be performed simultaneously with the test for sterility of the product to be examined.
4. Carry out a test as describe below under test for sterility of the product to be examined using exactly the same methods, except for the following modifications.
5. Sterility Test of Large Volume Products by Filtration Method

Membrane filtration:

1. After transferring the content of the containers to be tested (Sample content) to the membrane, add inoculums of a small number of viable microorganisms of each of the strains listed in Table No. 1 (NMT 100cfu) to the final portion of sterile diluents used to rinse the filter.
2. Perform a Growth Promotion Test as a positive control.
3. Incubate all the containers containing medium for NMT 5 days.

Direct Inoculation:

1. After transferring the contents of the container or containers to be tested to the culture medium, add an inoculum for a small number of a viable microorganisms (NMT 100 cfu) of each of the strains listed in Table No:1(NMT 100cfu) to the medium.
2. Perform a Growth Promotion Test as a positive control.
3. Incubate all the containers containing medium for NMT 5 days.

Interpretation:

1. If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control container without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. This test for sterility may then be carried out without further modification.
2. If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control containers without product, the product possesses antimicrobial activity that has not be satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the method suitability test.

Table-1 Test Microorganisms Suitable for Use in the Growth Promotion Test

Medium	Microorganism	Incubation (05 days)
		Strain	Temperature	Conditions
Fluid Thioglycollate	Staphylococcus aureus	ATCC 6538	32.5±2.5°C	Aerobic
	Pseudomonas aeruginosa	ATCC 9027	32.5±2.5°C	Aerobic
	Clostridium sporgenes	ATCC 11437	32.5±2.5°C	An aerobic
Soyabean Casein Digest	Bacillus subtilis	ATCC 6633	22.5±2.5°C	Aerobic
	Candida albicans	ATCC 10231	22.5±2.5°C
	Aspergillus niger	ATCC 16404	22.5±2.5°C

Procedure of Sterility Testing:

Follow the Gowning/ De-gowning procedure for Entrance / Exist in the aseptic area/Sterility suite as mentioned in the SOP

Membrane Filtration Method:

Opening of Containers:

The exterior surfaces of articles must be cleaned with an approved, filtered decontaminating agent (e.g:70% I.P.A Sterile) and gain access to the contents in an aseptic manner.

For Aqueous Solutions:

1. Draw sample from each of the article to be tested as per Table-2&3 and filter through the membrane filter with the aid of vacuum.
2. Rinse the membrane with 3 x 100ml Fluid A. (If product has antimicrobial properties, wash the membrane NLT 3 times with 3 x 100ml fluid D).
3. Aseptically remove the membrane filter from the filter holder, cut the membrane in two halves. Immerse the half portion of membrane into 100 ml of soybean casein digest medium and incubate at 20 to 25°C for 14 days.
4. Similarly immerse the other half of the membrane into 100ml fluid thioglycollate medium and incubate at 30 to 35°C for 14 days.

Negative Control:

1. A negative control is performed using chosen dilution and filter with add of pump. Or proceed with the test as described above for aqueous solutions.
2. Aseptically remove the membrane filter from the filter holder, cut the membrane in two halves. Immerse the half portion of membrane into 100 ml of soybean casein digest medium and incubate at 20 to 25°C for 14 days.
3. There must be no growth in both media (fluid thioglycollate or soybean casein digest medium). A failed negative control requires an investigation.

Observations & Interpretation of Results:

1. At interval during incubation period and its conclusion examine the media for macroscopic evidence of microbial growth.
2. If no evidence of microbial growth is found, the product to be examined complies with test for sterility.
3. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for cause unrelated to the product to be examined.

Table 2: Minimum Quantity to be used for each medium

Quantity per Container	Minimum Quantity to be used
Liquid(other than antibiotic):
Greater than 40ml, and not greater than 100ml	20ml.
Greater than 100ml	10% of the contents of container ,but not less than 20ml

Table3. Minimum Number of Articles to be tested in Relation to the number of Articles in the Batch

Number of Item in the Batch	Minimum Number of Items to be Tested for each Medium (unless otherwise justified and authorized)
Parenteral Preparations
Not more than 100 containers	10% or 4 containers, whichever is greater
More than 100 but not more than 500 containers	10 containers
For large-volume parenterals	2% or 20 containers, whichever is less
For large-volume parenterals	2% or 10 containers, whichever is less

Note: If the batch size is unknown, use the maximum number of items prescribed.

Investigation On Sterility Failure:

1. The test may be considered invalid only if one or more of the following condition are fulfilled.
2. The data of microbiology monitoring of sterility testing facility show a fault.
3. A review of the testing procedures used during test in question reveals a fault.
4. Microbial growth is found in the negative control.
5. After determination of the identity of the microorganism isolated from the test, the growth of this species may be ascribed unequivocally to fault with respect to the material and or the technique used in conducting the procedure.
6. Reason for positive result must be investigated as per the following.
7. Preserve the positive tube along with positive and negative control till to complete investigation.
8. Immediately streak the loop full suspension from sterility positive tubes on the pre incubated Tryptic Soya Agar plate in a duplicate.
9. If the suspension is streaked from Fluid Thioglycollate Medium, incubate the plates at 30 to 35⁰C for 48 hrs.
10. If the suspension is streaked from Soybean Casein Digest, incubate the plates at 20 to 25⁰C for 72 hrs.
11. After Incubation, observe the cfu and find out the colony by staining techniques or other biochemical techniques.
12. Simultaneously checks the results of microbiological monitoring taken while performing sterility test and during manufacturing and filling of the suspected product.
13. Identify the colony found during the plate exposure on testing date and also the microorganism found in preserved positive tube.
14. Also check the area cleaning and sanitization record for sterility testing room and manufacturing area.
15. Check the sterilization cycle details of the media, filtration assembly and utensils which is used for sterility testing including Sterilization temperature, pressure, holding time, chemical sterilization indicator.
16. Sterility Test of Large Volume Products by Filtration Method
17. Check the sterilization cycle details of suspected product.
18. Carefully review the manufacturing steps; special care has to be taken for filter integrity testing during filtration operation.
19. Simultaneously review the sterility testing procedure and training of sterility test operator.
20. Comply all the above data and find out the root cause.
21. If colony observed during Sterility testing is the same characteristics as found in positive tube, there are chances of contamination during testing.The test is declared to be invalid; it is repeated with same number of units as in the original test. If no evidence of microbial growth is found in the repeated test, the product complies with the test for sterility. If microbial growth is found in the repeat test, the product examined.

Sampling procedure for LVP (I n House)

Take 2 samples from 1st trolley, two samples from 4th trolley, two samples from 7th trolley, one sample from 2nd trolley, one sample from 3rd trolley, one sample from 5th trolley and one sample from 6th trolley.