General Test Procedure for Sterility Testing

PURPOSE

To lay down the procedure for performing the sterility testing of sterile finished goods and sterile raw materials.

APPARATUS

Conventional Method

1. Sterile Filtration Cups.
2. Sterile Manifold.
3. Scissors.
4. Forceps.
5. Sterile 0.45 µ, 47 mm edge hydrophobic membranes.
6. Vacuum pump.
7. Micropipette, Sterile Tips.
8. Suction flask.
9. Suction tube.
10. Sterile Vent filters.

Closed Method

1. Sterility test Compaq system
2. Dilutors.
3. Canisters.
4. Disposable Syringes.

REAGENTS

Sterile Fluid Thioglycollate Medium

Sterile Soyabean Casein Digest Medium.

0.1% Peptone.

Beta-lactamase.

PROCEDURE

Article to be Tested 

• The minimum Number of Articles to be tested in Relation to the Number of Articles in the Batch is described in table 1

Table 1

Number of Articles in the Batch	Number of Articles to be Tested
For Injections
More than 500 articles	2 % or 20 articles whichever is less.
For Antibiotic Solids Bulk and Blend Products
Aseptically remove a sufficient quantity of solids from the container mix to obtain a composite sample of not less than 10 g of solids.

Volume of Medium

• The volume of the medium used in the test should not be less than the volume indicated in Table 2.
• 100ml of Soya bean casein digest medium and 100ml of Fluid thioglycollate medium are prepared in Bottles for the Closed system and in test tubes for the Conventional method.0.1% of peptone in Bottles

Table: 2 Quantities of Articles for liquid Products.

Container Content (ml)	The minimum Quantity is taken from each container for each medium	Minimum Volume in ml for each medium for Membrane Filtration Method
10 to less than 50	5 ml	100

Table: 2 Quantities of Articles for Solid Products.

Container Content (g)	Minimum Quantity is taken from each container for each medium	Minimum Volume in ml for each medium for Membrane Filtration Method
200 – 300 mg	100 mg	200 ml
300 – 600 mg	200 mg	200 ml
> 600 mg	200 mg	200 ml

Opening of Articles

• Care must be exercised when opening an article so that the sample to be tested for sterility is not contaminated by the microorganisms present on the exterior of the container.
• The exterior surfaces of the ampoules, vials, and bottles must be cleansed with a suitable decontaminating agent and should be placed in an environment that prevents recontamination of the exterior surfaces.

Membrane Filtration Method

• Before starting the tests clean the LAF bench with 75 % filtered IPA.
• Place the sterilized manifold on the LAF bench and connect the tubing to the vacuum line through a collecting reservoir.
• Place the required number of filter holders on the manifold.
• Put the sterilized membrane/pre-sterilized in between the filter support with the help of sterile forceps and clamp it.

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Antibiotic Solids for Injections

Pharmacy Bulk Packages

• For the sterile bulk material, reconstitute about 6 gm of material in 200 ml sterile 0.1% Peptone previously inoculated with 0.6 ml of Beta-lactamase in a 500 ml conical flask.
• Shake or swirl to dissolve it completely.
• Transfer the entire contents into the filtration funnel.
• For sterile WFI ampoules, decontaminate the exterior of the ampoules using sterile 75% IPA, break the neck of glass ampoules and transfer the contents into the filtration funnel. In the case of plastic ampoules, withdraw the contents of the ampoules with the help of a sterile syringe & needle and transfer them into the filtration funnel.
• In case of sterile water for injections do not add Beta-lactamase.
• After the contents are filtered, rinse the membrane with three 100 ml portions of 0.1% Peptone containing 1.8 ml of Beta-lactamase.
• Remove the upper part of the funnel, and cut the membrane into two halves using sterile scissors and forceps.
• Aseptically transfer one half into fluid Thioglycollate medium and another half into Soyabean casein digest medium.
• Inoculate each medium tube with the validated quantity of Beta-lactamase having 1-lac units of Beta-lactamase enzyme. 0.6 ml for Bulk Active Pharmaceutical Ingredients and 2.0 ml for Sterile Dosage Forms.
• Each time when the Sterility Test is performed, include one tube of each medium as a negative control, which has been treated in a similar way as test samples except for the addition of a product to confirm the sterility of the medium.

Formulation

• From each of 20 containers aseptically transfer about 300 mg of solids into a sterile 200 ml conical flask containing 0.6 ml of Beta-lactamase, dissolve in 200ml of 0.1% Peptone and mix or reconstitute.
• Shake or swirl to dissolve the material completely.
• In case of sterile water for injections do not add Beta-lactamase.
• After the contents are filtered, rinse the membrane with three 100 ml portions of 0.1% Peptone containing 1.8 ml of Beta-lactamase.
• Remove the upper part of the funnel, and cut the membrane into two halves using sterile scissors and forceps.
• Aseptically transfer one half into fluid Thioglycollate medium and another half into Soyabean casein digest medium.
• Inoculate each medium tube with the validated quantity of Beta-lactamase having 1-lac units of Beta-lactamase enzyme. 0.6 ml for Bulk Active Pharmaceutical Ingredients and 2.0 ml for Sterile Dosage Forms.

Sterility by Steri Test

• Perform the testing of samples as per the SOP for the operation of the Sterility test.
• Before filtering the samples inoculate 0.6 ml of Beta-lactamase for sterile API and 2.0 ml of Beta-lactamase for sterile finished dosage forms containing 1.0 lac of Beta-lactamase into both the FTGM and SCDM bottles.
• Inoculate 0.6 ml / 2.0 ml of Beta-lactamase into the 0.1 % peptone solution bottles.

Precautions to be taken before/during the test for sterility

• A Qualified analyst shall always perform the sterility testing only.
• The blower of the garment cubicle should be always in running mode.
• Follow the gowning procedure as per the SOP correctly and efficiently.
• Always wear goggles during the test.
• All the media used for testing should be labeled properly for all the details such as

1. Name of the media
2. Batch number of sterilized media
3. Date of preparation
4. Prepared by.

• Use 70 % filtered IPA frequently to disinfect the gloves during the test.
• All the samples to be tested shall be arranged in a row and not in a bunch.
• All the materials placed in the LAF bench should be at a distance of 1.5 inches from the LAF grill to avoid the turbulence of filtered air.
• The samples reconstituted for testing should be immediately filtered without any delay to avoid false negative results.
• The forceps and the scissors used for cutting and inoculating the membranes should not be hot while holding the membrane.
• Frequently getting from the seat during the testing should be in less number.
• If any unforeseen observations e.g., sneezing, coughing, power failure or the membrane falling on the LAF bench etc. are noticed during the performance of the sterility test, abandon the testing and declare all the tests performed in that session as INVALID. Inform your Supervisor immediately about the incident.
• If the Temperature or differential pressure are not as per the acceptance criteria inform the maintenance department to take necessary action and after it is resumed go for sterility testing.
• Avoid excessive aeration of FTGM.

Incubation and Observation

• Incubate the Fluid thioglycollate medium tubes at 32.5° ± 2.5°C and Soyabean casein digest medium tubes at 22.5° ± 2.5°C for not less than 14 days.
• Observe the tubes on daily basis for any growth/turbidity during the incubation period and at the end of the incubation period and record the details
• Where the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be determined by visual examination transfer a portion of the medium ( NLT 10 ml ) to a fresh container of the same medium from the third to the seventh day after the test is started. Continue incubation of the original and the transferred container for a total of not less than 14 days from the original inoculation.
• If turbidity, precipitate or other evidence of microbial growth develops during incubation investigates the failure of the test as per the SOP.

Interpretation of Test Results

• The article meets the requirements of the test for sterility when no microbial growth is observed.

REFERENCE

USP

EUROPEAN PHARMACOPOEIA

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