Sterility Test in pharmaceutical plant 2023

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Sterility:

Sterility refers to the state of being free from all living microorganisms, including bacteria, viruses, and fungi. It is the absence of viable microorganisms in a specific environment, such as in a surgical field, laboratory equipment, or medical devices. Sterility can be achieved through various methods, including physical methods such as heat, radiation, and filtration, and chemical methods such as the use of disinfectants and sterilants. Sterility is critical in healthcare settings to prevent the transmission of infections and ensure patient safety

Sterility testing is designed to demonstrate the presence or absence of extraneous viable contaminating microorganisms in biological parenteral designed for human use.

1.0 OBJECTIVE:

To lay down a procedure for Sterility test

2.0 SCOPE:

The procedure is applicable for Sterility test in Microbiology  Labs.

3.0 RESPONSIBILITY:

3.1 Microbiologist / Trained person

3.1.1 To perform the written activities as per procedure.

3.1.2 To report any non compliance to the procedure

3.1.3 To prepare the trends and maintain a schedule of the activity as per procedure

3.2   Head Microbiology

3.2.1 To ensure the activities are performed as per procedure

3.2.2 To ensure the activities are performed as per the laid down schedules.

3.2.3 To investigate any non compliance to the procedure

4.0 ACCOUNTABILITY :

4.1 Head Quality Control & Quality Assurance

4.1.1 To ensure the procedure is implemented at their site

4.1.2 To investigate any non compliance to the procedure.

4.3.2 To verify investigation of any non compliance to the procedure.

5.0 PROCEDURE

5.1 Requirements:

  • Sterile test tubes
  • Sterile forceps
  • 2 µ filtered 70 % IPA
  • 45 µ sterilized filter membranes
  • Sterile lint free cloth/ mop
  • Sterile scissors/ blades/ cutter
  • Laminar Air Flow
  • Incubator (20 – 25 ºC)
  • Incubator (30 – 35 ºC)
  • Filtration assembly with vacuum pump

5.2 Media & Culture:

  • Soyabean Casein Digest Medium (SCDM)
  • Fluid Thioglycollate Medium (FTGM)
  • Diluent
  • Aspergillus brasiliensis (ATCC 16404)
  • Pseudomonas aeruginosa (ATCC 9027)
  • Staphylococcus aureus (ATCC 6538)
  • Environment isolates
  • Candida albicans (ATCC 10231)
  • Clostridium sporogenes (ATCC 11437)

5.3 Media/ sample/ articles transfer to sterility area:

  • Transfer the media/ samples/ articles to the sterility area through mobile LAF.
  • Don’t expose the samples under UV light at any point of time.
  • Use the sterilized items and accessories for sterility testing. Expose the sterile items and accessories for sterility testing to UV light for 15 – 20 minutes while transferring through mobile LAF.
  • Use filtration assembly, manifold, silicon tubing, scissors, and forceps sterilized by autoclave prior to use.
  • Check the seal integrity of sterilized items used for sterility testing. If wrapping of sterilized item is found torned/ compromised, do not use the same. Item need to be re-packed and re-sterilized.
  • Keep all the material related to sterility such as filtration assembly, sterile filters, disinfectants, scissors, and forceps in the mobile LAF.
  • From the mobile LAF, transfer the sample and accessories to the sterility LAF. In the LAF, each article is unwrapped and keep at its place e.g. manifold in the center of the sterility LAF, filtration assemblies to the manifold receptacle, sample to be tested should be placed on the left side of the LAF platform. Fix the vacuum flask to the vacuum pump through the silicon tubing. Media and other accessories like scissors and forceps on the right side of the LAF platform.
  • All the materials placed in the LAF bench should be at a distance of 1.5 inches from the LAF grill to avoid turbulence of filtered air.
  • Clean the outer surface of each article with 0.2 µ filtered 70 % sterile IPA before performing sterility testing.

5.4Sample quantity:

    • For sterility testing of in-process samples, 100 mL of sample shall be taken.
    • For sterility testing of sterile finished product, the sample quantity shall be taken as given in Table-I. The sample quantity per vial/ ampoule shall be taken as given in Table-II.
    • For sterility testing of pre-sterilized articles, sample quantity shall be taken as given in Table-III.
    • For sterility testing of depyrogenated vials/ ampoules, 20 samples shall be taken for both the media.
    • For sterility testing of sterile rubber bungs/ seals, 20 samples shall be taken for both the media.
    • For sterility testing of disinfectants, 200 mL of sample shall be taken for both the media.
    • For Filtered IPA, spray in a sterile bottle to get 200 mL sample.
    • For nitrogen and compressed air samples, 1000 liters air shall be purged in 200 mL sterile rinsing fluid. 200 mL sample shall be tested for sterility testing.
  • 5.5 Pre-requisites for Sterility Testing:
    1. Prepare the media and diluents as per respective SOP.
    2. Perform the Growth Promotion Test of prepared media as per respective SOP.
    3. Use 24 – 48 hours pre-incubated media for Sterility Testing.
    4. Transfer pre-incubated FTGM and SCDM from incubation room to the cooling zone through DPB. Then transfer the media to the sterility test room from cooling zone through mobile LAF.
    5. Transfer the diluents to sterile passage from cool zone directly. Then transfer the diluents to the sterility room from the sterile passage through mobile LAF.
    6. Enter in to the sterility area as per respective SOP.
    7. Arrange the samples to be tested on the LAF work station after surface disinfection with 0.2 µ filtered 70 % sterile IPA.
    8. Keep the accessories required for the sterility testing under LAF workstation after surface disinfection with 0.2 µ filtered 70 % sterile IPA.
    9. Remove the wrapping from all the sterilized materials inside the LAF cabinet.
    10. Before starting the sterility testing, check the resazurin layer of the fluid thioglycollate medium; it should not be more than 1/3rd of the total volume of medium in the tube/ vial.
    11. At the time of sterility testing of the product, mark the tube with the microbiology reference no. of product and media lot no. with permanent marker.

5.6 Method for Sterility Testing of Products:

A. Membrane Filtration Technique:

    1. Membrane filtration technique is generally used for injections, it generally depends on the nature of the sample to be tested i.e. which permits filtration.
    2. Carry out the procedure under aseptic condition using Class A LAF cabinet located in Class B clean room.
    3. Use membrane filters having a nominal pore size not greater than 0.45 µm whose effectiveness to retain microorganisms have been established.
    4. Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters for strongly alcoholic solutions.
    5. Switch “ON” the UV Light for 15-30 minutes.
    6. Collect the required quantity of sample mentioned in tables above and clean the outer surface and closure of ampoules/ vials with suitable decontaminating agent (0.2 µ filtered 70 % IPA), and keep in Class-A conditions to prevent recontamination of the outer surface (under LAF).
    7. Assemble the filtration assembly with manifold, in specified pattern i.e. connect the Vaccum pump with vacuum flask using a silicon tubing and manifold with the vacuum flask. Fit the filtration assembly on the manifold receptacle.
    8. Place the sterile filter paper of cellulose nitrate having pore size not greater 0.45µ and approx 50 mm diameter on the filter holder portion of the manifold receptacle assembly unit with the help of sterile forceps.
    9. Place the receptacle portion over membrane filter holder so that the membrane filter is trapped between the two portions of the assembly.
    10. Switch ON the vacuum pump and aseptically transfer 50-100 mL 0.1% sterile rinsing fluid to moisten the membrane filter.
    11. Cut the ampoule/ vial with sterile cutter/ opener and collect the whole content in the sterile container or directly pour the content of the ampoule/ vial into the filtration assembly.
    12. Filter the content with the aid of slow vacuum.
    13. Care must be exercised while opening the sample to be tested for sterility is not contaminated.
    14. The sample reconstituted/ dissolved for the testing should be immediately filtered without any delay avoids false negative results.
    15. Rinse the filter paper by filtering through it not less than three successive quantities, each of 100 mL of diluents, do not exceed a washing cycle of 5 times 100 mL per filter paper.
    16. In the case of liquid sample i.e. WFI, Sodium chloride and Sodium bicarbonate any other diluents not required for dissolving and rinsing purpose.
    17. After completion of filtration, cut the membrane filter into two equal parts with the help of sterile cutter/ scissor and insert one half of the membrane filter into the 100 mL of FTGM and other half into the 100 mL of SCDM aseptically.
    18. The membrane filter should be completely submerged in their respective media.
    19. Label the tubes with sample name, batch no., date of testing and date of completion along with the media lot no. with the signature of analyst performing the sterility.
    20. Perform the test along with Negative Control by rinsing the sterile membrane filter with 100 mL of sterile diluents.
    21. Incubate the same lot inoculated with test organisms to check growth promotion will be considered as FTGM tube at 30 – 35 °C and SCDM tubes at 20 – 25 °C for not less than 14 days.
B. Test For Negative Control:
      1. Perform the negative control test along with sterility test.
      2. Filter 100 mL of 0.1% peptone/ rinsing fluid through the filtration assembly.
      3. Similarly transfer membrane filter into SCDM and FTGM and incubate along with products (Diluent Negative Control).
      4. Keep one un-inoculated tube of SCDM and FTGM as a Media Negative Control along with the test for 14 days.
C. Test for Positive Control:
    1. Perform the positive control test in the Bio-safety cabinet of Micro Lab.
    2. For performing the positive control for the autoclaved lot of media, inoculate 10-100 cells of specified microorganisms in to the FTGM and SCDM media tubes.
    3. Incubate the positive control media tube along with test tubes to simulate the same incubation condition.
D. Direct inoculation technique:
    1. Transfer the quantity of the preparation to be examined as per Table-I and Table-II directly into the medium in such a way that the volume of the product is not more than 10% of the volume of medium, unless otherwise prescribed.
    2. If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium.
    3. When it is necessary to use a large volume of the product it may be preferable to use a concentrated culture medium prepared in such a way that it takes account of the subsequent dilution. Where appropriate, the concentrated medium may be added directly to the product in its container.

E. Observations and Result Reporting:

      1. During the incubation period and at its conclusion, examine the media for evidence of microbial growth/ turbidity on daily basis.
      2. Maintain the record of sterility testing as per Annexure-I.
F. Interpretation of results:
    1. If no evidence of microbial growth is found, the sample/ article examined complies with the test for sterility when compared with the negative diluting fluid control.
    2. If evidence of microbial growth is found, the sample/ article examined does not comply with test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the examined product.
    3. In such case an OOS related to sterility testing should be declared and investigation shall be initiated.
G. The test is considered as invalid under the following conditions:
      1. The data of the environmental monitoring of the sterility testing facility shows a fault.
      2. A review of the testing procedure used during the test in question reveals a fault.
      3. Microbial growth is found in the negative control.
      4. After determination of the identity of the microorganisms isolated from the test containers showing microbial growth, the growth of this species or these species may be ascribed without any doubt to faults with respect to the materials or the technique used in conducting the sterility test procedure.

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