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.
5.4.1.1 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
8.3.1.1 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.
8.3.1.2 Aseptically remove the aluminum foil seal with a
sterile decapper and allow it to dry.
6.3.1.3 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.
6.3.1.4Transfer 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.
6.3.1.5 Aseptically transfer half contents for each of 20
ampoules into 200 ml of diluting fluid A contained in a screw capped
flask.
6.3.1.6 For 1ml volume ampoules whole contents will be used.
6.3.1.7 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:-
8.3.2.1 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.
8.3.2.2 Using a sterile spatula aseptically transfer approx.
6 gm of bulk into a flask containing diluting fluid.
8.3.2.3 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].
8.3.2.4 Swirl gently
without splashing the contents of the flask.
8.3.2.5 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:
8.3.3.1 Set up the Millipore
filtration system under LFH.
8.3.3.2 Remove the outer covering
of aluminum foil in the hatch and then transfer the apparatus
under LFH with the inner covering.
8.3.3.3 Before the start of the test remove the inner
covering of aluminum foil and set the assembly to make it ready for filtration
process.
8.3.3.4 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.
8.3.3.5 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.
8.3.3.6 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.
8.3.3.7 Filter the
sample by using vacuum pump.
8.3.3.8 Rinse the filter
once with 200 ml of sterile peptone water.
8.3.3.9 Filter 200 ml of
peptone water containing same quantity of pencillinase as used in case of test
sample and treat it as negative control.
8.3.3.10 Remove the valve on each filter system after each
filtration and at the end of the entire procedure.
8.3.3.11Open the caps of media flasks as FTM & Soyabean Casein Digest
Medium / TSB and heat their lips properly.
8.3.3.12 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.
8.3.3.14 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
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·
Material/Product Sterility
Test Report
·
Sterility Test Data.
·
Area Monitoring of
Sterility Testing Room
10. References:
10.1 United States
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
|
Received
(Current)
|
Returned
(Obsolete)
|
|
1
|
Quality Control
Department
|
|
|
|
2
|
Quality Management
Department
|
|
|
12
Revision History:-
Date Changes
N/A
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