SOP for Sterility Test

This Document describes (Standard Operating procedure) SOP for Sterility Test

SOP for Sterility Test

I. Purpose & Scope:

• The purpose of this SOP is to lay down the procedure for Sterility testing by using Membrane filtration Method and by Direct inoculation method.    
• This Standard operating procedure shall be applicable for Sterility testing of Sterile API, Finished products intended for use as injectables and any other sterile material.

II. Responsibilities:

• All Quality control personnel shall be responsible to follow and implement this SOP.

III: Introduction and procedure:

Introduction:

Sterility test is intended for detecting the presence of viable microorganisms in sterile preparations. The test is based on the principle that, if microorganisms are placed in a medium, which provides nutrients with water and kept at a favorable temperature, the organisms will grow and their presence shall be indicated by turbidity in the originally clear medium.

General Requirements :

• Sterile Manifold holder assembly
• SS Filtration cup
• Vacuum pump
• Sterile Forceps and Scissors
• Sterile Garments
• Sterile cutter
• Sterile 0.45µ Membrane filter papers
• 2 µ Syringe filters with syringe
• Sterile 0.1% Peptone
• Sterile Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM)

Note: Perform the Sterilization of Accessories and medium in Autoclave. Equipment and Accessories shall be sterilized at HPHV cycle (At 121.5°C for 30 min) and liquid/Agar Medium should be sterilized at Standard cycle-1 (At 121.5°C for 20 min).

Media preparation for sterility Testing : 

Use following media for sterility testing :

Soybean casein digest medium.

Fluid Thioglycollate medium.

Fluid-A (0.1% w/v Peptone water).

• Prepare the media as per respective SOP and sterilize them.
• The media used for test should pass the Growth Promotion Test, carried out before or in parallel with the test.
• If the Growth Promotion Tests carried out in parallel with the test is failed, the media used in the test stands invalid and the test shall be repeated.

Growth promotion test of medium : 

• During testing, Carry out growth promotion test or Fertility testing of Fluid Thioglycollate medium (FTGM) using 10 to 100 CFU of viable microbial inoculums of following organisms:

1. Staphylococcous aureus  (ATCC 29737)
2. Pseudomonas aeruginosa (ATCC 9027)
3. Clostridium sporogenus (ATCC 11437)

• During testing , Carry out growth promotion test or Fertility testing of Soybean casein digest medium (SCDM) using 10 to100 CFU of viable microbial inoculums of following organisms:

1. Bacillus subtilis (ATCC 6633)
2. Aspergillus brasiliensis (ATCC 16404)
3. Candida albicans (ATCC 10231)

• Incubate the tubes of Soybean casein digest medium at 20^o to 25^o C and of Fluid thioglycollate medium at 30^o to 35^o C for not more than 3 days in case of bacteria & not more than 5 days in case of fungi. Record the result in Sterility testing report. GPT passes if growth is exhibited by the test organism within specified period.

Sample preparation: 

• Great care must be exercised when opening an article, so that the sample to be tested for sterility is not contaminated by microorganisms present on exterior of the container.
• Minimum number of articles to be tested in relation to the number of articles in the batch as follow

Number of items in the batch	Minimum number of items to be tested
1) For Finished product: If quantity of batch size is more than 500 containers	2% or 20 containers whichever is less.
2) For Solid bulk Raw material	100% sampling

 

• Minimum quantity to be tested per vial in relation to the quantity per container as follow:

Quantity per container	Minimum quantity to be tested per vial
1) Finished Product: If Quantity per container is 300 mg to 5gram	300 mg
2) Solid bulk	Minimum about 6 gram

• Performing the sterility testing procedure as per given in point 6

 Sterility testing by membrane filtration method : 

• For sterility testing take representative samples of the whole batch. Make entry of sample in Sterility inward register, refer Annexure-II.
• Ensure that all equipment, media and samples to be tested are ready and suitable before actual test and before entering in sterility testing area.
• Wipe the external surface of sample articles; Sterility media like SCDM and FTGM individually with filtered 70% IPA solution. Keep them in clean SS drum/tray, and then transfer it to the sterility room.
• Enter into Sterility area as per entry procedure of respective SOP.  Unload the sterilized material from double door autoclave. Transfer all the accessories and prepared media to sterility testing room through clean dynamic pass box and keep it in LAF.
• Expose the Soybean casein digest agar (SCDA) plates at specified location for environment monitoring of the sterility testing area, during sterility testing.
• Unload the sterilized accessories from double door autoclave and transfer them to sterility area through dynamic pass box.
• Check the differential pressure of working LAF. Also check the temperature and humidity of the sterility room.
• Aseptically connect the filtration assembly in LAF. SS Filtration cup shall be fitted with SS manifold holder, properly in laminar air flow cabinet.
• At first, aseptically place the sterile 0.45µ membrane filter in the filtration cup by using sterile forcep. Transfer small quantity (approx.50ml) of sterile fluid-A (0.1% peptone) to filtration cup to moisten the membrane filter, allow it to filter by applying vacuum.
• For filled vials, aseptically open the vials using sterilized cutter/scissor and transfer the defined quantity of content from 20 vials in sterile 100 ml Fluid-A (0.1% Peptone water), mix the whole content properly and transfer entire content to filtration cup.
• For raw material, add desired quantity of sample (about 6gm) in Sterile 100 ml 0.1% Peptone water and transfer entire content to Filtration cup.
• Apply vacuum to filter the product immediately.
• After completion of sample filtration, give three rinses of 100 ml (3 X 100 ml) sterile 0.1% Peptone water to the membrane filter.
• Release the vacuum after completion of filtration, Lift the membrane carefully with the help of sterile forceps and aseptically cut the membrane filter into two halves using sterile forceps and scissor. Immerse one half into 100 ml sterilized Soybean casein digest medium (SCDM) and another half into 100 ml sterilized Fluid Thioglycollate Medium (FTGM). Label the medium tubes with Name of media, batch number, and date of analysis.
• Simultaneously prepare a negative control by filtering 100 ml of sterile 0.1% Peptone water instead of sample. Cut the membrane into two halves with sterile SS scissor and transfer one half in SCDM and another half in FTGM tube.
• If product is antibiotic in nature i.e Cephalosporin group then use the neutralizing enzyme (Beta-lactamase-1 supplement) to neutralize the antimicrobial activity of the products. At first reconstitute the enzyme vial by sterile water. Filter the enzyme by 0.2µ Nylon Syringe filter by using sterile syringe.
• After product filtration, give three rinsing to membrane filter by 3 X100 ml sterile 0.1% Peptone water, it shall be performed along with beta lactamase enzyme (add 0.2 ml of beta lactamase having minimum potency 1000 IU/Vial).
• At every rinse of 100 ml 0.1% Peptone water, add 0.2 ml of beta lactamase enzyme having minimum potency 1000 IU/Vial. After each rinse of Fluid-A, along with 0.2 ml of beta lactamase enzyme, give minimum one minute of contact time and then start the filtration. Proceed the activity as mentioned above in point 6.12.
• After completion of work, transfer all the test tubes and all the exposed plates to respective incubators. Transfer the used accessories to washing area. Before exit from sterility area, perform the Personnel monitoring.
• Incubate the tested Soybean casein digest medium tubes along with negative control at 20°C-25°C and Fluid Thioglycollate medium Tubes along with negative control at 30°C-35°C for not less than 14 days.
• Perform the positive control of SCDM and FTGM in Microbiology testing room in LAF as mentioned in Point 4
• Observe all tubes daily for presence of growth, in terms of turbidity till the completion of incubation period.
• Record the observations of the sterility test in Annexure-I.

Sterility testing by Direct Inoculation Method: 

Some of the sterile materials or items cannot be tested for sterility by Membrane filtration method. Alternatively, direct inoculation method can be used for sterility testing of such samples. In this method the sample articles are directly inoculated in medium aseptically and incubated at specified temperature and time.

Sterility test of Sterile Gloves: 

• After receiving the sample, make an entry in Sterility testing inward register, Refer Annexure-II.
• Ensure that all accessories, media and samples to be tested are ready before entering in sterility testing area.
• Use appropriate bottles or flasks of suitable size to prepare media for sterility test of sterile gloves.
• Transfer the samples and required material such as presterilized media and accessories to sterility testing.
• Enter into Sterility area as per respective SOP. Unload the sterilized material from double door autoclave. Transfer all the accessories and prepared media to sterility testing room and keep it in LAF.
• Expose the Soybean casein digest agar (SCDA) plates at specified location for environment monitoring of the sterility testing area, during sterility testing.as per SOP.
• Aseptically open the one pack of sterile gloves sample; it contains a pair of gloves.
• Cut the one glove, into small pieces by using sterile scissor and sterile forceps.
• Aseptically immerse the pieces of glove into sterile SCDM media bottle.
• The remaining glove is to be treated in same way and the pieces are to be immersed in FTGM media bottle.
• After completion of work, transfer all the test tubes and all the exposed plates to respective incubators. Transfer the used accessories to washing area. Before exit from sterility area, perform the Personnel monitoring.
• Incubate the tested Soybean casein digest medium tubes along with negative control at 20°C-25°C and Fluid Thioglycollate medium Tubes along with negative control at 30°C-35°C for not less than 14 days.
• Perform the positive control of SCDM and FTGM in Microbiology testing room in LAF.
• Observe all tubes daily for presence of growth, in terms of turbidity till the completion of incubation period.
• Record the observations of the sterility test in Annexure-I.
• Frequency: As and when sample received.

Interpretation of sterility test Results: 

• At intervals during the incubation period, and at its conclusion, examine the contents of all the tubes for the evidence of microbial growth.
• Sample shall meet the requirements of sterility if :
• Growth Promotion Test of the medium used in the sterility test shows early and copious growth (Maximum within 3 days in case of Bacteria & 5 days in case of Fungi).
• Negative Control test media shows no growth and remains clear till the end of incubation.
• No growth is observed in the sample test tube of both the media.
• If the growth in the product sterility test is observed, quarantine the batch, and initiate complete investigation.

Investigation of sterility Failure: 

• In case of sterility test failure observed, quarantine the complete batch and initiate the investigation accordance with Quality Assurance Dept.

Investigate following parameter of production and Quality control department.

Production Department:

Environmental Conditions of manufacturer of the products :

1. Environment monitoring results
2. Personnel monitoring results
3. Surface monitoring results
4. Pressure differentials between classified areas
5. Temperature and humidity of rooms
6. Training of persons who has manufactures the batch
7. Machine interventions

Details of following test :

1. Integrity test
2. Sterilization records
3. Sealing quality (Integrity of vials)

Quality Control (Microbiology) Department :

• Room count on the day of sterility test. Environment monitoring results of Sterility area, LAF and Personnel monitoring results.
• Training of persons who has performed sterility test.
• Sanitization and cleaning of sterility testing room.
• Sterilization condition of equipment/media used in sterility.
• Check whether microbial growth is observed in Negative control.
• If the investigation proves beyond doubt, that the test is a laboratory induced false positive, then repeat the test using same number of sample as in original test.
• If no evidence of growth in the repetition observed then sample passes the test for sterility declaring first test invalid.
• If no assignable cause is found in Laboratory and Manufacturing investigation. As well as growth in the repetition testing is observed then Quaqlity Assurance Dept. shall carry out failure investigation and impact assessment. Quality Assurance Head shall take a decision of Approval or Rejection of the batch.

IV: Annexures :

Annexure I         :  Sterility testing report

Annexure II    :  Sterility testing inward register

 

Annexure- I

Sterility testing report

 

1. I) Sample and Incubation Details :

Name of Product / Material		Date of Testing
Batch Number		A. R. Number
Lot number of SCDM		Lot number of FTGM
Lot number of 0.1% Peptone		Neutralizing enzyme details
Incubator I.D used for SCDM (20-25°C)		Incubator I.D used for FTGM (30-35°C)
Done By		Date of completion

    

1. II) Observation :

Incubation Day	Date	SCDM	FTGM	Negative Control	Observed By	Checked By
1st Day
2nd Day
3rd Day
4th Day
5th Day
6th Day
7th Day
8th Day
9th Day
10th Day
11th Day
12th Day
13th Day
14th Day
15th Day *

* Column to be used for recording observation if 14^th day is holyday.

H = Indicates Holiday, – ve  Indicates no growth(sterile),  +ve  Indicates growth

 

 III) Observations of Positive control and Negative controls :

 

1. Soybean Caseine digest medium :

Name of Organisms	Positive control	Observed On/By

       

Negative control: ______________________

 

1. Fluid thioglycollate Medium :

Name of Organism	Positive control	Observed On/By

 

Negative control:  ______________________

 

Conclusion: The analyzed sample found Satisfactory/Not Satisfactory.

 

Analysed By:                                                                                  Checked By:

Date:                                                                                                Date:

 

Annexure II

Sterility testing inward Register

 

  

Name of Product	Batch. No	A. R. No	Analysis done on	Analysis Done By	Observed  By	Checked By	Remark