AMV Protocol for Sterility by Filtration Method
Analytical Method Verification Protocol for Sterility by Filtration Method
|Sr. No.||Title||Page No.|
|—||Table Of Contents|
|2.0||Qualification Team and Responsibilities|
|3.0||Introduction, Objective and Scope|
|4.0||Performance Qualification Pre-requisites|
|6.0||Performance Qualification Procedure|
|8.0||Re qualification Criteria|
|9.0||Deviation, Change Control and Corrective action|
|10.0||Summary, Conclusion and Recommendation|
|Prepared by||Quality Control|
|Approved by||Quality Assurance|
2.0 Qualification Team and Responsibilities:
|Quality control department shall be responsible for-
1. Documentation of data during the qualification study.
2. Operating the system/equipment as per written standard operating procedures.
Review of protocol and report.
|Production||Production department shall be responsible for-
1. Necessary support during qualification.
2. Provide the sample for Performance Qualification study.
|Quality Assurance||Quality Assurance department shall be responsible for-
1. Review and Approval of Protocol and Report
2. Review the data documented during equipment qualification and ensure that it meets all the specification requirements.
3. Compilation of summary report.
4. Review of the data documented during Performance qualification and ensures compliance with all specification.
5. Investigation, verification and approval of any change control generated during the Performance qualification study.
6. Approval of Performance qualification study.
3.0 Introduction, Objective and Scope:
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 microorganisms 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 if the membrane filtration method is used.
The Objective of this report is to establish documented evidence that the methodology for the method verification report of sterility test by Membrane filtration for process and finished product, when performed as per the standard Operating Procedures.
Procedure laid down by this report shall be applicable for sterility test of product at microbiology laboratory of Abc Pharma Ltd.
The purpose of this activity is to check the suitability of the sterility test method and to establish documentary evidence that the sterility test method is capable of giving reproducible and consistent results.
4.0 Performance Qualification Pre-Requisites:
All the Equipments and Instruments used for the validation study shall be Qualified and Calibrated.
4.1 Primary Requirement
First three consecutive batches of finished product.
Dilutions of Challenge Organisms containing 10-100 Cfu/ml (For list of organisms given ahead).
Betalactamase / Penicillinase
Sterilized Filtration Assembly
Sterilized Filtration cup.
Glass Test tube 38 x 200mm – Rack and SS Stand.
Sterilized Forceps and Scissors.
Edge hydrophobic cellulose nitrate filter 0.45 μ pore size.
Calibrated Incubators set at temperature 32.5 ± 2.5oC and 22.5 ± 2.5oC.
Isopropyl Alcohol 70 % v/v.
4.2 Media Required
Glass test tube 38 x 200mm containing 100ml medium of Fluid Thiogycollate Medium.
Glass test tube 38 x 200mm containing 100ml medium of Soyabean Casein Digest Medium.
Peptone Solution 0.1% w/v for Sample dissolution and rinsing.
Peptone Solution 0.1% w/v fluid A containing Betalactamase /Penicillinase Fluid A.
Buffered Sodium Chloride Peptone solution pH 7.0 for culture dilutions.
4.3 Cultures Required
|Sr. No||Name of the Challenge Organism||ATCC No|
4.4 List of Documents
Ensure the availability of the associated document viz. Environment monitoring, Entry and exit procedure for sterility area, Preparation and storage of culture suspension SOP and qualification protocols etc.
5.1 Record the detail of reference documents in Annexure-I.
5.2 Protocol reference: USP chapter <71>, sterility test, European Pharmacopoeia (Ph. Eur.): sterility.
6.0 Performance Qualification Procedure
6.1 Growth Promotion Tests of the Medium
6.1.1 This test shall be performed to prove that the test tube containing media when comes in contact with the medium does not inhibit the growth of the microorganism.
6.1.2 The organism selected for the GPT study shall be as described in Table-I.
6.1.3 The challenge incoulum level shall be 10-100 Cfu/ml.
6.1.4 The quantification of the inoculum shall be done as per SOP on “Preparation and storage of microbial culture suspension” as per SOP No.
6.1.5 Copy of culture suspension record shall be attached along with the report.
6.1.6 The inoculation of the challenge microorganisms shall be done under BSC in the Culture handling room of the Microbiology Laboratory.
6.1.7 The selection of the challenge organism shall as per Table- II for both the medium,
SCDM or FTGM.
6.1.8 Prepare the soyabean casein digest medium and fluid thioglycollate medium as per SOP on
“Preparation and usage of sterile media” as SOP No.
6.1.9 The growth promotion test shall be performing as per current SOP on “Media Growth Promotion,
Inhibitory and Indicative Properties” as per SOP No.
6.1.10 Glass test tube containing Fluid Thioglycollate medium shall be incubated at 32.5 + 2.5oC and
Soybean Casein Digest medium at 22.5 + 2.5oC.
6.1.11 The negative control of Fluid Thioglycollate Medium test tube shall be incubated at 32.5 + 2.5oC for maximum 3 days. Soybean Casein Digest medium test tube shall be incubated at 22.5 + 2.5oC for
maximum 5 days.
6.1.12 The growth promotion properties record as per Format on “Growth promotion / indicative /
inhibitory properties of media as a Format No
|S. No||Challenge organism||Medium||Incubation temperature||Maximum Incubation time|
|01||St. aureus (ATCC 6538)||FTGM||30-35oC||3 days|
|02||Cl. sporogenes (ATCC 19404)||FTGM||30-35oC||3 days|
|03||P. aeruginosa (ATCC 9027)||FTGM||30-35oC||3 days|
|04||Environment Isolate||FTGM||30-35oC||3 days|
|05||C. albicans (ATCC 10231)||SCDM||20-25oC||5 days|
|06||B. subtilis (ATCC 6633)||SCDM||20-25oC||3 days|
|07||A.brasiliensis (ATCC 16404)||SCDM||20-25oC||5 days|
|08||Envionment Isolate||SCDM||20-25oC||3 days|
6.2 Sterility Test
6.2.1 The all required material transferred through the dynamic pass box with UV treatment.
6.2.2 Microbiologist enter into the sterility area as per SOP on “Entry and exit procedure for Sterility area” as per SOP NO.
6.2.3 This test shall be performed to confirm the sterility of the three consecutive batches of the product tested for Bacteriostasis and Fungistasis test.
6.2.4 Sterility testing of the batches shall be performed for the batches along with the Bacteriostasis and Fungistasis test.
6.2.5 6g of the sample shall be tested for the sterility test. (<5 g from each of 20 containers, transfer about 300mg of solids, into 500ml conical flask / glass bottle dissolved in about 200 ml of peptone water).
6.2.6 The total rinse volume of each test tube shall be of 3 x 100 ml for each membrane.
6.2.7 Test tube containing Fluid Thioglycollate Medium with membrane shall be incubated at 32.5 + 2.5oC and Soybean Casein Digest with membrane at 22.5 + 2.5oC for a period of 14 days.
6.2.8 After performed the test microbiologist take self-personal monitoring during exit time as per SOP on “Environment monitoring”
6.2.9 The personal monitoring record shall be attached with validation report.
6.2.10 Microbiologist/s shall observe the incubated test tube daily and results in sterility test report Format No.
6.2.11 If no evidence of microbial growth is found, the product sample being examined passes the tests for sterility.
6.2.12 If the presence or absence of microbial growth cannot be readily determined by visual examination, 14 days after the beginning of incubation, 1mL of the medium shall be transferred to fresh sterilized test tube of the same medium and incubated further.
6.2.13 The original test tube and transfer test tube shall be incubated for not less than 4 days.
6.2.14 If no evidence of microbial growth is found, the product sample being examined passes the tests for sterility.
6.2.15 If evidence of microbial growth is found, the product examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product examined.
6.2.16 In case, if growth is observed in the test tube, the results shall be communicated to QA Head for the investigation and the method validation shall be performed for another set of three consecutive batches after getting clearance from QA.
6.3 Bacteriostasis and Fungistasis Test
6.3.1 This test shall be performed to confirm that the membrane in the test tube after filtering the product is rinsed with the sufficient volumes of the rinsing fluid which is capable of removing any traces of the product by subjecting to the challenge organisms.
6.3.2 6g of the sampled product shall be tested for the bacteriostasis and fungistasis test for each microorganism separately- as given in table -I. Inoculate the two 100 ml fluid A bottle with 10 – 100 cfu of each microorganisms written in table –I.
6.3.3 This study shall be performed by using diluents in which product is dissolved.
6.3.4 When test is performed by adding Betalactamase 200 IU shall be added in 100 ml of media after sterilization.
6.3.5 The test tube containing media (SCDM and FTGM), validation product, rinsing fluid containers, reconstitution bottles and other related test aids shall be transferred to the culture handling room.
6.3.6 Sanitize the BSC bench with 0.2μ filtered 70%IPA.
6.3.7 Transfer the sterilized membrane cups, sterilized 0.45μ hydrophobic edge membranes, rinsing fliuid container, reconstitution bottle and other related test aids shall be transferred to the culture handling room bio safety cabinet.
6.3.8 Assemble the filtration unit (filtration cup on unit consisting of glass receptacle between which a properly supported membrane in placed) whole set should be sterilized and testing done under aseptic condition.
6.3.9 Use the filter paper having pore size not greater than 0.45μ, hydrophobic edge membrane and diameter approximately of 47 mm.
6.3.10 Assemble the sterilized filter set to the filtration unit under bio safety cabinet.
6.3.11 Aseptically place the sterilized/per-sterilized membrane using the sterilized forceps in the base of the filtration cup.
6.3.12 After fixing the sterilized/per-sterilized membrane, mount the top portion of the filtration cup.
6.3.13 Transfer the 20-50 ml of sterilized peptone water to the membrane and filter using the vacuum pump for wetting purpose.
6.3.14 Transfer the sterile product 6 gram to a bottle containing 200 ml of sterile peptone.
6.3.15 Wait till the product sample dissolves completely.
6.3.16 Transfer the entire contents and simultaneously filter the sample with the help of pump immediately without any hold up of the product in the filtration unit.
6.3.17 Transfer 3 x 100ml of sterilized peptone to the membrane with the help of pump. This volume is for rinsing the membrane for neutralization. (In case of with betalactamase test – fourth washing shall be given with 200 IU containing betalactamase in 100 ml peptone water). For last or fourth washing use 100 ml of peptone water containing 10 -100 cfu of each microorganism.
6.3.18 Transfer this filter to as per specified in table-I to respective media and after this activity, remove the filter assembly.
6.3.19 For using Betalactamase repeat the procedure as point no. 6.3.10 to 6.3.17. Now transfer the filter paper to test tube of respective media as per table-I containing Betalactamase.
6.3.20 Repeat all this procedure for rest of all microorganisms.
6.3.21 Incubate the Soyabean Casein Digest Medium tube with membrane at 22.5 + 2.5oC and Fluid Thioglycollate Medium tube with the membrane at 32.5 + 2.5oC for a period of NMT 5 and 3 days respectively.
6.3.22 Microbiologist/s shall observe the incubated test tube with membrane daily and document the results in Annexure-I.
6.3.23 The negative control of Fluid Thioglycollate Medium tube shall be incubated at 32.5 + 2.5oC and Soybean Casein Digest medium tube shall be incubated at 22.5 + 2.5oC for a period of 14 days along with the negative product control.
6.3.24 The Bacteriostasis and Fungistasis test study shall be performed only after routine sterility testing of the batches and shall be performed as a separate activity in the culture handling room.
6.3.25 The test tube having growth shall be decontaminated as per SOP on “Decontamination and disposable of used media”
6.3.26 In case, if growth is not observed in the test tube, the Bacteriostasis and Fungistasis test shall be repeated with modification in the rinsing fluid volume.
6.4 Stasis Test
6.4.1 This test is performed to ensure that media is still fertile after the completion of incubation period of 14 days and there is no inhibitory effect of drug substance filtered through the membrane.
6.4.2 The sterility tested test tube of all the three batches after the 14days incubation period shall be subjected to the growth promotion test by adding 10-100Cfu/ml of challenge organisms.
6.4.3 The activity shall be performed under biosafety cabinet in culture handling room of the controlled area of the Microbiology laboratory.
6.4.4 Clostridium sporogenes ATCC 19404 for Fluid Thioglycollate Medium test tube and Candida albicans ATCC 10231 for Soybean Casein Digest Medium shall be chosen as the challenge organism.
6.4.5 10-100 Cfu/ml of the quantified suspension shall be inoculated in both media test tube by micropipette.
6.4.6 Glass test tube containing Fluid Thioglycollate Medium shall be incubated at 32.5 + 2.5oC and Soybean Casein Digest media containing test tube at 22.5 + 2.5oC for a period of not more than 3 days for bacteria and 5 days for fungus.
6.4.7 Microbiologist/s shall observe the incubated test tube daily and document the results in Annexure-II.
6.4.8 If growth is not apparent within 3 days of bacteria and 5 days of fungus, the test is considered invalid.
6.4.9 The Invalid stasis tests shall be repeated once.
6.4.10 If growth is not obtained at the second attempt the sterility test method shall be modified and revalidated.
6.5 Gram staining/Lactophenol cotton blue staining.
6.5.10 The growth obtained test tube shall be subjected to Gram staining/Lactophenol cotton blue to confirm the growth of the organism challenged.
6.5.11 The test shall be performed as per SOP on “Gram staining” (SOP No. ), Lactophenol cotton blue staining (SOP No.)” respectively.
6.5.12 The gram staining/Lactophenol cotton blue results shall be documented in Annexure-III.
6.5.13 In case if the organism do not match with the challenge organism, the source of the cross contamination shall be identified and the bacteriostasis and fungistasis test shall be repeated for the concerned microorganism.
6.6 Test Precautions
6.6.1 During testing material movement of material should be very slowly under biosafety cabinet.
6.6.2 Filter paper set mount the top of filtration cup safely.
6.6.3 After dissolving the product completely filter the solution immediately.
7.0 Acceptance criteria
7.1 Growth promotion tests of the medium
7.1.1 The test tube medium should show growth within 3 days for bacterial cultures and within 5 days for yeast and molds cultures.
7.2 Sterility testing of the product
7.2.1 The batches tested for sterility for the validation batches should pass the sterility tests by showing no turbidity of the medium.
7.3 Stasis Test
7.3.1 If growth is not apparent within 3 days for bacteria and 5 days for fungi, the test is considered invalid.
7.3.2 Invalid stasis tests shall be repeated once.
7.3.3 If growth is not obtained at the second attempt the sterility test method shall be modified and revalidated.
7.4 Bacteriostasis and Fungistasis test.
7.4.1 In the bacteriostatic and fungistasis test, the medium in the challenged test tube should show growth in the form of turbidity of the medium within 3 days for bacterial cultures and within 5 days for yeast and molds cultures.
8.0 Re- Qualification Criteria
8.1 Failure to meet the requirement will require the change in procedure and if in any case the procedure is changed revalidation will be performed.
8.2 If no growth observed in bacteriostasis and fungistasis test after three washing then validation activity shall be performed with increased number of washings.
8.3 A new vendor is approved for betalactamase and filter paper.
8.4 A new product is manufactured.
8.5 The manufacturing process is changed.
8.6 Any modification of room or equipment.
9.0 Deviation, Change Control and Corrective Action:
If during study any deviation is observed in procedure same shall be handled as per SOP on “Handling of Deviation”, (SOP No.) and its impact shall be assessed before implementation.
If protocol is required to be revised, Change control shall be taken as per SOP on “Change control”, (SOP No.) Record the deviation and change control in Annexure- I.
10.0 Summary, Conclusion and Recommendation:
Summary, conclusion and recommendation shall be recorded as per Annexure – I.
11.0 Final Approval:
Final approval shall be given as per Annexure I.
|SOP||Standard Operating Procedure|
|SCDM||Soyabean Casein Digest Medium|
|FTGM||Fluid Thioglycollate Medium|
|ATCC||American Type Culture Collection|
|Cfu||Colony Forming Unit|
END OF PROTOCOL