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GENERAL TEST PROCEDURE (GTP) Page 1 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

REVISION SUMMARY

Version
Date Revised Reason for Revision
Number
GTP 031 - 00 17/05/2000 First Issue
GTP 031 - 01 05/09/2001 Second Issue
GTP 031 - 02 01/07/2002 Third Issue
GTP 031 - 03 01/01/2003 Fourth Issue
GTP 120 - 04 01/05/2003 Renumbering of GTP and Format Changed, Method
Enhanced
GTP 120 - 05 01/10/2003 Incorporation of investigation for failure SOP
GTP 120 - 06 10/01/2004 The Word USP is removed from the topic as this procedure is
common for both USP and EUROPEAN
PHARMACOPOEIA.
GTP 120 - 07 04/01/2005 The format of the GTP is changed as per SOP QA 030-00 and
the procedure is revised.

1.0 PURPOSE
1.1 To lay down the procedure for performing the sterility testing of sterile finished goods and
sterile raw materials.

2.0 APPARATUS

Prepared By Checked By Approved By


Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 2 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

2.1 Conventional Method.


a) Sterile Filtration Cups.
b) Sterile Manifold.
c) Scissors.
d) Forceps.
e) Sterile 0.45 µ, 47 mm edge hydrophobic membranes.
f) Vacuum pump.
g) Micropipette, Sterile Tips.
h) Suction flask.
i) Suction tube.
j) Sterile Vent filters.
2.2 Closed method.
a) Steritest Compaq system
b) Dilutors.
c) Canisters.
d) Disposable Syringes.

3.0 REAGENTS
3.1 Sterile Fluid Thioglycollate Medium
3.2 Sterile Soyabean Caesin Digest Medium.
3.3 0.1% Peptone.
3.4 Beta lactamase.
4.0 PROCEDURE
4.1 ARTICLES TO BE TESTED.
Minimum Number of Articles to be tested in Relation to the Number of Articles in the Batch are
described in table 1
Table 1
Number of Articles in the Batch Number of Articles to be Tested

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Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 3 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

For Injections
More than 500 articles 2 % or 20 article which ever is less.
For Antibiotic Solids Bulk and Blend Products
Aseptically remove a sufficient quantity of solids from the container mix to obtain a
composite sample not less than 10 g of solids.

4.2 Volume of Medium


4.2.1 The volume of the medium used in the test should not be less than the
volume indicated in Table 2.
4.2.2 100ml of Soya bean casein digest medium and 100ml of Fluid thio
glycollate medium is prepared in Bottles for Closed system and in test
tubes for Conventional method.0.1% of peptone in Bottles
Table: 2 Quantities of Articles for liquid Products.
Container Minimum Quantity taken Minimum Volume in ml for each
Content (ml) from each container for medium for Membrane Filtration
each medium Method
10 to less than 50 5 ml 100

Table: 2 Quantities of Articles for Solid Products.


Container Minimum Quantity taken Minimum Volume in ml for each
Content (g) from each container for medium for Membrane Filtration
each medium Method
200 – 300 mg 100 mg 200 ml
300 – 600 mg 200 mg 200 ml
> 600 mg 200 mg 200 ml

4.3 Opening of Articles


4.3.1 Care must be exercised when opening an article so that the sample to be

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Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 4 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

tested for sterility is not contaminated by the microorganisms present on


the exterior of the container.
4.3.2 The exterior surfaces of the ampoules, vials and bottles must be cleansed
with a suitable decontaminating agent and should be placed in an
environment that prevents recontamination of the exterior surfaces.

4.4 Membrane Filtration Method.


4.4.1 Before starting the tests clean the LAF bench with 75 % filtered IPA .
4.4.2 Place the sterilized manifold on the LAF bench and connect the tubing to
the vacuum line through a collecting reservoir.
4.4.3 Place the required number of filter holders on the manifold.
4.4.4 Put the sterilized membrane / pre sterilized in between the filter support
with the help of a sterile forceps and clamp it.

4.5 Antibiotic Solids for Injections


4.5.1 Pharmacy Bulk Packages.
4.5.1.1 For the sterile bulk material, reconstitute about 6 gms of
material in 200 ml sterile 0.1% Peptone previously inoculated
with 0.6 ml of Beta lactamase in a 500 ml conical flask.
4.5.1.2 Shake or swirl to dissolve it completely.
4.5.1.3 Transfer the entire contents into the filtration funnel.
4.5.1.4 For sterile WFI ampoules, decontaminate the exterior of the
ampoules using sterile 75% IPA, break the neck of glass
ampoules and transfer the contents into the filtration funnel.
In case of plastic ampoules, withdraw the contents of the
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Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 5 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

ampoules with the help of a sterile syringe & needle and


transfer into the filtration funnel.
4.5.1.5 In case of sterile water for injections do not add Beta
lactamase.
4.5.1.6 After the contents are filtered, rinse the membrane with three
100 ml portions of 0.1% Peptone containing 1.8 ml of Beta
lactamase.
4.5.1.7 Remove the upper part of the funnel, and cut the membrane in
two halves using sterile scissors and forceps.
4.5.1.8 Aseptically transfer one half into fluid Thioglycollate medium
and other half into Soyabean casein digest medium.
4.5.1.9 Inoculate each medium tube with the validated quantity of
Beta lactamase having 1-lac units of Beta lactamase enzyme.
0.6 ml for Bulk Active Pharmaceutical Ingredients and 2.0 ml
for Sterile Dosage Forms.
4.5.1.10Each time when the Sterility Test is performed, include one
tube of each medium as negative control, which has been
treated in the similar way as test samples except the
addition of product to confirm the sterility of the medium.
4.6 Formulation:
4.6.1 From each of 20 containers aseptically transfer about 300 mg of solids
into a sterile 200 ml conical flask containing 0.6 ml of Beta lactamase,
dissolve in 200ml of 0.1% Peptone and mix or reconstitute.
4.6.2 Shake or swirl to dissolve the material completely.
4.6.3 Perform the procedure as outlined in steps number 4.5.1.5 to 4.5.1.9.

4.7 Sterility by Steri test


4.7.1 Perform the testing of samples as per the SOP number QC261 for the
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GENERAL TEST PROCEDURE (GTP) Page 6 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

operation of Steri test.


4.7.2 Before filtering the samples inoculate 0.6 ml of Beta lactamase for sterile
API and 2.0 ml of Beta lactamase for sterile finished dosage forms
containing 1.0 lac of Beta lactamase into both the FTGM and SCDM
bottles.
4.7.3 Inoculate 0.6 ml / 2.0 ml of Beta lactamase into the 0.1 % peptone
solution bottles.

4.8 Precautions to be taken before / during the test for sterility


4.8.1 A Qualified analyst shall always perform the sterility testing only.
4.8.2 The blower of the garment cubicle should be always in running mode.
4.8.3 Follow the gowning procedure as per the SOP correctly and efficiently.
4.8.4 Always wear goggles during the test.
4.8.5 All the media used for testing should be labeled properly for all the details
such as
4.8.5.1 Name of the media
4.8.5.2 Batch number of sterilized media
4.8.5.3 Date of preparation
4.8.5.4 Prepared by.
4.8.6 Use 70 % filtered IPA frequently to disinfect the gloves during the test.
4.8.7 All the samples to be tested shall be arranged in a row and not in bunch.
4.8.8 All the materials placed in the LAF bench should be at a distance of 1.5
inches from the LAF grill to avoid turbulence of filtered air.
4.8.9 The samples reconstituted for testing should be immediately filtered
without any delay to avoid false negative results.
4.8.10 The forceps and the scissors used for cutting and inoculating the
membranes should not be hot while holding the membrane.
4.8.11 Frequently getting from the seat during the testing should be in less
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Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 7 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

number.
4.8.12 If any unforeseen observations e.g., sneezing, coughing, power failure or
the membrane fell on the LAF bench etc. are noticed during the
performance of sterility test, abandon the testing and declare all the tests
performed in that session as INVALID. Inform your Supervisor
immediately about the incident.
4.8.13 If the Temperature or differential pressure are not as per the acceptance
criteria inform the maintenance department to take necessary action and
after it is resumed go for sterility testing.
4.8.14 Avoid excessive aeration of FTGM.

4.6.2 Incubation and Observation:


4.6.2.1 Incubate the Fluid thioglycollate medium tubes at 32.5° ± 2.5°C and Soyabean
casein digest medium tubes at 22.5° ± 2.5°C for not less than 14 days.
4.6.2.2 Observe the tubes on daily basis for any growth / turbidity during the incubation
period and at the end of incubation period and record the details
4.6.2.3 Where the material being tested renders the medium turbid so that the presence
or absence of microbial growth cannot be determined by visual examination
transfer a portion of the medium ( NLT 10 ml ) to a fresh container of the same
medium from the third to the seventh day after the test is started. Continue
incubation of the original and the transferred container for total of not less than
14 days from the original inoculation.
4.6.2.4 If turbidity, precipitate or other evidence of microbial growth develops during
incubation investigate the failure of the test as per the SOP number QC238.
4.6.3 Interpretation of Test Results
4.6.3.1 The article meets the requirements of the test for sterility when no microbial
growth is observed.

Prepared By Checked By Approved By


Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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GENERAL TEST PROCEDURE (GTP) Page 8 of 8

Title Sterility Testing Effective Date 04/01/05

GTP Number GTP 120-07 Review Date 03/01/07

5.0 REFERENCE
5.1 USP 27
5.2 EUROPEAN PHARMACOPOEIA-2005.

END OF DOCUMENT

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Name G.SRIKANTH N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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