1.     Purpose:-                         

            This procedure describes the method for sterility test
2.     Scope:-

This test is performed to ensure the sterility of the sterile raw materials, sterile products,    sterile packaging materials and all other materials which are claimed as sterile.

3.     HSE Statement:-

            Proper gowning technique should be followed.

4.     Responsibilities:-

                               i)      Manager Quality Control is responsible to ensure that procedure & formats are followed entirely as approved.
                             ii)        Microbiologist is responsible to perform the test.
                           iii)        Lab attendant is responsible to assist the  microbiologist for the preparation of test
5. Materials Required:-
5.1 Membrane Filters:
   Membrane filters should be compatible to the materials to be tested.
Pore size is 0.45 μm and the diameter 47 or 50 mm. They are individually packed and sterilized. The edge of the filters is hydrophobic. Cellulose nitrate filters are normally used in sterility test.
 5.2 Sterility Testing Apparatus:
A suitable 3 or 6 manifold membrane filter unit (e.g. Millipore, Sartorius or equivalent properly wrapped and sterilized) is used with detachable funnels, filter holders and funnel covers.
5.3  Medias:
5.3.1  Fluid Thioglycollate Medium  (FTM)
               Sterilized and pre-incubated for 2-5 days at 30-35OC temperature, dispensed in 100 ml quantities
               in suitable glass containers.
       5.3.2 Soybean Casein Digest Medium (SCDM) (TSB)
Sterilized and pre-incubated for 2-5 days at 20-25OC or at room temperature, dispensed in 100 ml quantities in suitable glass containers.
5.4 Diluting Fluids:
 5.4.1   Fluid ‘A’ (Sterilized and Pre-Incubated at 22-30oC for 2-5 Days)
Dissolve 1g of peptic digest of animal tissue in water to make 1 liter, filter or centrifuge to clarify if necessary, and adjust to a pH of 7.1and dispense into containers and sterilize using a validated process.    Preparation for Penicillin and Cephalosporin
Aseptically add to above preparation, if necessary a quantity of sterile Pencillinase enzyme sufficient to inactivate any residual activity on the membranes after solution of test specimen has been filtered. 
 5.4.2 Fluid ‘D’ (Sterilized)  
This fluid is used to inactivate preservatives and when product contains oil or lecithin. It contains 0.1% polysorbate 80 and 0.1% peptone.
5.5  General Requirements:-
       5.5.1 Sterilized medium size spatulas for measuring 6 grams in case of bulk powder and small size spatulas for measuring 300 mg for filled units.
                5.5.2 Spatulas and scissors wrapped as pair wise in aluminum foil, dry heat at 200 oC for a maximum of 2 hours.                                                                                                                                                                                                                                       
       5.5.3 Disinfectant:  70% alcohol filtered, 5% phenol, 3% Savlon, 5-10 % Chlorine bleach solution, Quaternary Ammonium compounds or equivalent.
5.5.4      Sterilized lint free wipes.
5.5.5      Suitable sized sterilized S/S containers with S/S covers for dumping of 20 units to be tested.
5.5.6        S/S bowls for disinfectant.
5.5.7        Filled unit opening device.
5.5.8        Sterilized de-capper or vial opener in case of vials or, sterilized ampoule cutter.
5.5.9        Sterile complete uniform including masks, surgical gloves etc.
5.5.10    Vacuum pump.
5.5.11    Filtration flask (2 liter capacity).
5.5.12    Autoclave-able rubber tubing, silica gel column and 2 liter filtration flask to the sterility testing unit.
5.5.13    Petri dishes for air count and gloves per procedures.
      5.5.14 Sterile glass or disposable syringes for liquid testing (optional).
      5.5.15 Gas burner.
 All materials for testing are sent inside the sterility testing suite through the hatch. Materials are wrapped in aluminum foil and sterilized either by dry heat (200˚C for NLT 2.0 hours) or by autoclaving. Remove the outer wrapping in the hatch and transfer the material inside under LFH. Materials that are not sensitive to UV light exposure are kept there for at least 30 minutes to minimize the outside microbial bioburden. Perform growth promotion test of all medias to be used in the test as per SOP.

6. Definitions:-

6.1  Sterility test       
     Within the strictest definition of sterility, an article is deemed to sterile when there is complete absence of viable microorganisms. Absolute sterility cannot be practically demonstrated because it is technically unfeasible to prove a negative absolute.
6.2 Sterility a test that critically assesses whether a
sterilized pharmaceutical product is free from contaminating microorganisms'.
6.3 Membrane Filtration Method
 This procedure is applicable to all the sterile materials, which when in liquid form can pass through the membrane filter of pore size of 0.45 um. For example Eye drops, Ampoules, Powder vials and sterile bulks.

6.4 Fluid Thioglycollate Medium (FTM):-
FTM is primarily intended for the cultivation of anaerobic bacteria.

6.5 Soybean Casein Digest Broth (Tryptic Soy Broth) (TSB):-
TSB is suitable for the culture of both fungi and aerobic bacteria.
6.6 Peptone Water:-
  Water-soluble digests or hydrolysates of proteins that are used in the preparation of culture media. Peptone water is used as diluting and rinsing fluid in sterility testing.
6.7 Negative Control: - To verify testing conditions a negative control is performed using the chosen diluents in place of test preparation. There must be no growth of microorganisms.

7.0Flow Chart:-

8.0 Procedure:-

8.1 Adopt gowning for sterility test as per SOP.
8.2 Sample Requirements:
                                      Collect the sample of product under test as per SOP.
8.3 Sample Preparation:
8.3.1 Finished Stage Products Decontaminate vials / ampoules by submerging in sterile 5% phenol solution or any other suitable approved disinfectant solution contained in sterile glass beaker for at least 30 minutes. Place the container in the hatch. Transfer the sample container under LFH after wiping with sterile cotton duster containing approved disinfectant. Remove the vials from the container under LFH and allow drying under LFH. Aseptically remove the aluminum foil seal with a sterile decapper and allow it to dry. Using a sterile spatula remove (flat side) the rubber plug in one jerk keeping the vials at an angle of 45Oto the laminar flow bench. aseptically powder sample from each 20 filled vials into 200 ml of diluting fluid A (Peptone Water) contained in a screw capped flask, as follows:
·         Whole contents of products having filled weight of 250 mg.
·         300 mg of products having filled weight of 500 mg to 1.0 gm. Aseptically transfer half contents for each of 20 ampoules into 200 ml of diluting fluid A contained in a screw capped flask. For 1ml volume ampoules whole contents will be used. Replace the cap gently on the flask and screw the cap tight. (Extensively flame with a Bunsen burner the lip of the flask prior to dumping of the sample & before replacing the cap].
8.3.2 Bulk Products:- Wipe the outside of the sample bag with a duster soaked in a suitable disinfectant solution and place in the hatch 20 minutes prior to testing. Before transferring the samples bag into testing room disinfectant again and place it under LFH and allow to dry. Using a sterile spatula aseptically transfer approx. 6 gm of bulk into a flask containing diluting fluid. Replace the cap carefully and screw the cap tight. (Extensively flame with a Bunsen burner the lip of the flask prior to dumping of powder and before replacing the cap]. Swirl gently without splashing the contents of the flask. Set the preparation aside for a minimum of one hour to permit complete inactivation of penicillin and for the sample to dissolve completely.
8.3.3 Sample Filtration: Set up the Millipore filtration system under LFH. Remove the outer covering of aluminum foil in the hatch and then transfer the apparatus  
             under LFH with the inner covering. Before the start of the test remove the inner covering of aluminum foil and set the assembly to make it ready for filtration process. Centre a sterile membrane filter, aseptically, using a fine sterile flat tipped forceps on each of the filter holder to be used and clamp the funnel tight. Connect the filtration assembly to filtrate collection flask as well as to vacuum pump. The collecting vessel along with the tubing is steam sterilized for 30 minutes, properly rapped in aluminum foil. Unscrew the flask containing prepared sample. Flame the lip of the flask and pour the entire contents into the filtration funnel without dripping the sample down the side of the flask. Filter the sample by using vacuum pump. Rinse the filter once with 200 ml of sterile peptone water. Filter 200 ml of peptone water containing same quantity of pencillinase as used in case of test sample and treat it as negative control. Remove the valve on each filter system after each filtration and at the end of the entire procedure. the caps of media flasks as FTM & Soyabean Casein Digest Medium / TSB and heat their lips properly. Remove the filter funnel and pick the filter using a sterile flat tipped forceps, from the base of the filter holder.
8 .3.3.13 Cut the filter in to two equal halves with the help of sterile scissors and dip one piece in FTM and one piece in Soyabean Casein Digest Medium / TSB. Proceed the same way for all other samples.
8.4 Environmental Monitoring During Test
8.4.1    Expose plate of Tryptic Soy Agar (TSA) under LFH for the whole duration of the test in space between the analyst and the test apparatus.
8.4.2    One plate in sterility testing suit.
8.4.3    One plate in buffer area and one plate in change room.
8.4.4    Gloves monitoring of the analyst is to be performed at the end of the test.
8.5 Incubation
8.5.1    Remove all the media out in the lab, Incubate FTM at 30 – 35 ºC & Soyabean Casein Digest Medium / TSB at 20 – 25 ºC for 14 days.
8.5.2    Incubate all tested plates at 30 – 35 ºC for a minimum of 48 hours.
8.6 Positive Control
Positive control of media is performed only on receiving of new lot or changed lot of media in order to check the efficacy of testing media. Run a positive control by adding one ml       
of different bacterial / mold cultures, available in the lab, to a flask of diluting fluid A containing powder equivalent to 20 vials of 250 mg or volume equal to 20 ampoules. Separate sample for all products is taken and proceeded in the same way. Proceed the filtration in the same way as described above, except that glass filtration assembly is used instead of S/S Millipore filtration assembly in order to avoid any contamination in routine sterility test. Also filtration process is carried out in bacterial sub culturing lab under safety cabinet instead of sterility testing suite. Use the same media and incubation temperature and time as used for test.
8.7 Interpretation of Sterility Test Results
 During incubation period observe the test media flasks daily for the evidence of turbidity due to any microbial growth. Product is sterile if no evidence of growth is found throughout the incubation period. When microbial growth is observed and confirmed microscopically, the article does not meet the requirements of the sterility test. However if the microbial growth can be without a doubt ascribed to faulty aseptic techniques or materials used in conducting the sterility testing procedure, the test is invalid and must be repeated. Repeat the test on same number of units as already tested in previous test.

9.    Records
·         Material/Product Sterility Test Report
·         Sterility Test Data.
·         Area Monitoring of Sterility Testing Room

10. References:
10.1   United States Pharmacopoeia 35. 
10.2 Guidelines on the Test Methods for Environmental Monitoring for Aseptic Dispensing Facilities, Produced by: A Working group of the Scottish Quality Assurance Specialist Interest Group, February 2004. (For All Classes at Rest and Operational B, C and D)

11. Distribution:-

This SOP has to be distributed in below mentioned Departments:-

Sr. NO
Distributed to
Quality Control Department

Quality Management Department


12 Revision History:-
Date                                        Changes