1.0 Objective
To lay down Standard Operating Procedure is to provide guidelines for sterility testing by Direct Inoculation Method.2.0 Scope
This procedure is applicable for the products manufactured at the location which can not be tested by Membrane Filtration Method.3.0 Responsibility
Implementation: Microbiologist4.0 Accountability
Execution: Manager - QC
Review & Approval : AGM - QA / QC
5.1.2 Sample for Intermediates: Randomly collect 16 pre-sterilized bottle samples to be tested for sterility from LVP bottle pack machine separately in such a way that each bottle should represent each cavity of the mold of bottle pack machine.
5.1.3 Transfer the samples to QC department in clean plastic crates.
5.1.4 Wipe the sample article individually with 70% IPA solution and keep in a clean stainless steel tray marked with Product Name, Batch No and Lot No, and then transfer the samples to the sterility room through clean pass box for performing sterility.
5.1.5 Prepare the media tubes (FTM and SCDM) as per the SOP for preparation of culture media, dispense 100-100 ml quantity in test tubes & plug them (for oily products add 10-10 g/l Polysorbate 80 to FTM & SCDM). Sterilize both the media at 121ºC and 15 psi pressure for 20 minutes as per SOP for Media Sterilization by Autoclaving.
5.1.6 After autoclaving Label the tubes with Name of Media, Media Batch No. and pre-incubate the media tubes at appropriate temperature i.e. SCDM tubes at 20 to 25ºC whereas FTGM tubes at 30 to 35ºC for 24 - 48 hrs before subjecting them for sterility operations.
5.1.7 Autoclave Dress & a conical flask with a cotton plug in an S.S Container separately at 121ºC temperature and 15 psi pressure for 30 minutes as per SOP. Autoclave 100 ml of WFI at 121ºC 15 psi pressure for 20 minutes for negative control.
5.1.8 Transfer the pre-incubated sterile media tubes, SCDA plates, sterile swabs to the sterility test room through pass box.
5.1.9 Enter in sterility room as per the Entry / Exit procedure for Sterility Room.
5.1.10 Start the LAF as per SOP for operating Instruction for LAF.
5.1.11 Wipe out all samples to be tested for sterility with 70% IPA solution.
5.1.12 Before starting sterility test, expose the SCDA plates as specified locations throughout the testing as per SOP.
5.1.13 Check the Manometer reading of working LAF and check the temperature as well as humidity of the sterility room
5.1.14 Manometer reading of working LAF chamber pressure should be between 08 – 15mm of water. Temperature reading of the sterility room should be 27ºC ± 2ºC.
5.1.15 Cut the tip of bottle or ampoule with sterile SS blade in front of the gas burner and immediately transfer the whole content of the ampoule for SVP to sterile conical flask aseptically. Similarly, transfer the content of all 20 ampoules to the sterile conical flask & mix.
5.1.16 Now take 10 ml sample from the conical flask & transfer to 100 ml sterile FTM tube. Similarly, transfer 10 ml sample to 100 ml sterile SCDM tube.
5.1.17 Label both the tubes with the product name, B. No, lot No., date of testing, Completion date & Tested by.
5.1.18 Simultaneously prepare a negative control by transferring 10-10 ml sterile WFI to 100-100 ml sterile FTM & SCDM tubes and label both the tubes as a Negative control.
5.1.19 Simultaneously prepare a chamber control during the sterility take two tubes, one is SCDM & other one is FTM tube, unplug the cotton plug of the tube and expose in LAF during sterility, after completion of sterility replug the tubes and then incubate the tubes as a chamber control.
5.1.20 After completion of work transfer all inoculated media through hatch box and then transfers all the equipment and exposed plates to microbiology analysis section.
5.1.21 Immediately move from the sterility area as per exit procedure specified SOP for Entry-Exit Procedure for Sterility Room.
5.1.22 Incubate the FTM tubes at 30ºC – 35ºC and SCDM tubes at 20ºC – 25ºC for 14 days. The incubation period for terminally sterilized products is not less than 7 days and 14 days for aseptically filled products as per IP. If the Product is as per USP, BP, incubation period is 14 days for both terminally sterilized as well as for aseptically filled products.
5.1.23 Start the LAF of Microbiology Analysis room as per SOP and prepare four positive Control tubes by inoculating aseptically 10 to 100 cfu in FTM tubes with S. aureus NCIM 2079, P. aeruginosa NCIM 2200, B. subtilis NCIM 2063 and environmental flora. Similarly prepare three SCDM positive control by inoculating approx 10 to 100 cells separately with C. albicans NCIM 3471, A. niger NCIM 1196 and environmental flora. Incubate FTM positive control tubes at 30 – 35ºC for 3 days & SCDM positive control tubes at 20 – 25ºC for 5 days.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria
5.2.2 If no evidence of growth observed in any of the tube the product to be examined for the test complies with the test for sterility.
5.2.3 The test is not valid unless the negative control shows negative till at end of incubation, and positive control shows growth within specified incubation period.
5.2.4 Destroy all the positive controls after confirmation of growth in both FTM and SCDM tubes.
5.2.5 If evidence of microbial growth is found in any of the tubes, investigate the cause of its failure as per SOP for Sterility test failure investigation. If investigation report concludes that test is invalid, repeat the test with 20 units.
5.2.6 If no evidence of growth is found in the repeat test the product examined complies with the test for sterility. If evidence of microbial growth is found in the repeat test the product examined does not comply with the test for sterility.
5.3.2 Any material used in the sterility operation or inside sterility room should be autoclaved properly.
5.3.3 The tip of sample bottles/ vials should be heated properly in front of gas burner before cutting with a sterile heated cutting blade.
5.3.4 Clean the waste solution reservoir immediately after sterility operations and mop the outer surface of the reservoir with filtered 70% IPA with sterile mopper.
Related: Incubation of Petridishes in Inverted Position
QA: Quality Assurance
QC: Quality Control
NA: Not Applicable
IPA: Isopropyl Alcohol
SVP: Small volume Parenterals
LVP: Large volume Parenterals
FTM: Fluid Thioglycollate Medium
SCDM: Soybean casein digest medium
IP: Indian Pharmacopoeia
BP: British Pharmacopoeia
USP: United States Pharmacopoeia
Review & Approval : AGM - QA / QC
5.0 Procedure
5.1 Direct Inoculation Method
5.1.1 Sample for Finished Products: Collect the samples to be tested for sterility. Out of the whole sample, randomly select 20 articles of each lot of batch for both LVP and SVP terminally sterilized products and for aseptically filled products.5.1.2 Sample for Intermediates: Randomly collect 16 pre-sterilized bottle samples to be tested for sterility from LVP bottle pack machine separately in such a way that each bottle should represent each cavity of the mold of bottle pack machine.
5.1.3 Transfer the samples to QC department in clean plastic crates.
5.1.4 Wipe the sample article individually with 70% IPA solution and keep in a clean stainless steel tray marked with Product Name, Batch No and Lot No, and then transfer the samples to the sterility room through clean pass box for performing sterility.
5.1.5 Prepare the media tubes (FTM and SCDM) as per the SOP for preparation of culture media, dispense 100-100 ml quantity in test tubes & plug them (for oily products add 10-10 g/l Polysorbate 80 to FTM & SCDM). Sterilize both the media at 121ºC and 15 psi pressure for 20 minutes as per SOP for Media Sterilization by Autoclaving.
5.1.6 After autoclaving Label the tubes with Name of Media, Media Batch No. and pre-incubate the media tubes at appropriate temperature i.e. SCDM tubes at 20 to 25ºC whereas FTGM tubes at 30 to 35ºC for 24 - 48 hrs before subjecting them for sterility operations.
5.1.7 Autoclave Dress & a conical flask with a cotton plug in an S.S Container separately at 121ºC temperature and 15 psi pressure for 30 minutes as per SOP. Autoclave 100 ml of WFI at 121ºC 15 psi pressure for 20 minutes for negative control.
5.1.8 Transfer the pre-incubated sterile media tubes, SCDA plates, sterile swabs to the sterility test room through pass box.
5.1.9 Enter in sterility room as per the Entry / Exit procedure for Sterility Room.
5.1.10 Start the LAF as per SOP for operating Instruction for LAF.
5.1.11 Wipe out all samples to be tested for sterility with 70% IPA solution.
5.1.12 Before starting sterility test, expose the SCDA plates as specified locations throughout the testing as per SOP.
5.1.13 Check the Manometer reading of working LAF and check the temperature as well as humidity of the sterility room
5.1.14 Manometer reading of working LAF chamber pressure should be between 08 – 15mm of water. Temperature reading of the sterility room should be 27ºC ± 2ºC.
5.1.15 Cut the tip of bottle or ampoule with sterile SS blade in front of the gas burner and immediately transfer the whole content of the ampoule for SVP to sterile conical flask aseptically. Similarly, transfer the content of all 20 ampoules to the sterile conical flask & mix.
5.1.16 Now take 10 ml sample from the conical flask & transfer to 100 ml sterile FTM tube. Similarly, transfer 10 ml sample to 100 ml sterile SCDM tube.
5.1.17 Label both the tubes with the product name, B. No, lot No., date of testing, Completion date & Tested by.
5.1.18 Simultaneously prepare a negative control by transferring 10-10 ml sterile WFI to 100-100 ml sterile FTM & SCDM tubes and label both the tubes as a Negative control.
5.1.19 Simultaneously prepare a chamber control during the sterility take two tubes, one is SCDM & other one is FTM tube, unplug the cotton plug of the tube and expose in LAF during sterility, after completion of sterility replug the tubes and then incubate the tubes as a chamber control.
5.1.20 After completion of work transfer all inoculated media through hatch box and then transfers all the equipment and exposed plates to microbiology analysis section.
5.1.21 Immediately move from the sterility area as per exit procedure specified SOP for Entry-Exit Procedure for Sterility Room.
5.1.22 Incubate the FTM tubes at 30ºC – 35ºC and SCDM tubes at 20ºC – 25ºC for 14 days. The incubation period for terminally sterilized products is not less than 7 days and 14 days for aseptically filled products as per IP. If the Product is as per USP, BP, incubation period is 14 days for both terminally sterilized as well as for aseptically filled products.
5.1.23 Start the LAF of Microbiology Analysis room as per SOP and prepare four positive Control tubes by inoculating aseptically 10 to 100 cfu in FTM tubes with S. aureus NCIM 2079, P. aeruginosa NCIM 2200, B. subtilis NCIM 2063 and environmental flora. Similarly prepare three SCDM positive control by inoculating approx 10 to 100 cells separately with C. albicans NCIM 3471, A. niger NCIM 1196 and environmental flora. Incubate FTM positive control tubes at 30 – 35ºC for 3 days & SCDM positive control tubes at 20 – 25ºC for 5 days.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria
5.2 Observation and Interpretation of Results
5.2.1 Visually examine the media tubes daily to its conclusion for macroscopic evidence of microbial growth.5.2.2 If no evidence of growth observed in any of the tube the product to be examined for the test complies with the test for sterility.
5.2.3 The test is not valid unless the negative control shows negative till at end of incubation, and positive control shows growth within specified incubation period.
5.2.4 Destroy all the positive controls after confirmation of growth in both FTM and SCDM tubes.
5.2.5 If evidence of microbial growth is found in any of the tubes, investigate the cause of its failure as per SOP for Sterility test failure investigation. If investigation report concludes that test is invalid, repeat the test with 20 units.
5.2.6 If no evidence of growth is found in the repeat test the product examined complies with the test for sterility. If evidence of microbial growth is found in the repeat test the product examined does not comply with the test for sterility.
5.3 Precaution
5.3.1 Sample bottles/ vials should be wiped properly with filtered 70% IPA before transferring them to sterility room and also inside the sterility room before sterility operations. Pass box should also be cleaned properly with filtered 70% IPA solution.5.3.2 Any material used in the sterility operation or inside sterility room should be autoclaved properly.
5.3.3 The tip of sample bottles/ vials should be heated properly in front of gas burner before cutting with a sterile heated cutting blade.
5.3.4 Clean the waste solution reservoir immediately after sterility operations and mop the outer surface of the reservoir with filtered 70% IPA with sterile mopper.
Related: Incubation of Petridishes in Inverted Position
6.0 Abbreviation
SOP: Standard Operating ProcedureQA: Quality Assurance
QC: Quality Control
NA: Not Applicable
IPA: Isopropyl Alcohol
SVP: Small volume Parenterals
LVP: Large volume Parenterals
FTM: Fluid Thioglycollate Medium
SCDM: Soybean casein digest medium
IP: Indian Pharmacopoeia
BP: British Pharmacopoeia
USP: United States Pharmacopoeia
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