MICLAB 060 Sample

Standard Operating Procedure
Title: Micro Laboratory Procedure for Sterility Testing

______________________________________________________________________________________

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6. Repeat Testing of Sterile Products..................................................................................................... 7
7. Media Check after Sterility Testing (Stasis Test)................................................................................ 8
8. Validation and Revalidation of Sterility Test Methods......................................................................... 8
9. Appendix 1 - Flowchart of Sterility Test results - Interpretation and Retests.......................................11
10. Summary of Changes.......................................................................................................................12

Procedure
1. Obtaining of Samples
1.1. Sampling
Production personnel conduct all sampling in the following areas. Details of these sampling
procedures are outlined in MICLAB 095.
Terminally Sterilised Products
Samples are to be selected randomly from throughout the autoclave load.
Aseptically Filled Products
Samples are to be selected from the beginning, middle and end of each batch, plus the first
units filled after any prolonged downtime (greater than one hour). Samples are to be of
saleable standard, not rejects.
1.2. Sample Size required for Initial Sterility Testing
Determine the sample size of the product type and batch size.
2. Setting up a Testing Session
2.1. Checking the testing requirements for each product code
Every product sampled must be documented properly in details when setting up a test
session.
This will indicate what tests are required for each product code. Always cross-check the
BPN on the sample container against the sample ID sheet.
2.1.1. Parametric Release
Not all terminally sterilised products require sterility testing. Check for Parametric
Release status of any product code.
2.1.2. Sterility Test
These products require sterility testing. A standard sterility testing session is
comprised of 4 products and a sterile control to be membrane filtered and any
number of direct inoculations along with a suitable sterile control.
2.1.3. Bacterial Endotoxin
Check if a product requires Bacterial Endotoxin testing and also by which test
method, either gel clot or KCA. If the product requires Bacterial Endotoxin testing,
separate these samples from the sterility samples. Ensure that these samples are
labelled with the Product Name & strength, batch, code and place these in the
receptacle for the type of Endotoxin test required.
2.1.4. Biological Assay
If a biological assay is required separate the samples from the Sterility samples.
Ensure these samples are labelled with the Product Name, batch and code and are
given or communicated to the technician responsible for Assay testing.
2.2. Known Sterile Controls
______________________________________________________________________________________

Incubate the TSB and FTM bottles as described below in point 3.6 Incubation.
Secondary Transfer Process
After not less than 14 days of incubation and working within the sterility test LAF,
aseptically transfer the following:
- 1 mL of the initial FTM media into a new bottle of 100mL FTM, and,
- 1 mL of the initial TSB media into a new bottle of 100 mL TSB.
Continue the incubation of the initial media and transfer media bottles as follows:
FTM Not less than 4 days @ 322C.
TSB Not less than 4 days @ 222C.
Examine the media for growth at intervals during the incubation period. If no
evidence of growth is found the batch meets the requirements for the Test of
Sterility.
3.2. Agar Plates
One irradiated Letheen Agar plate and one irradiated Nutrient Agar plate are to be taken into
the Sterility Test room for every sterility test session.
The Nutrient Agar plate is to be left exposed on the Laminar Flow bench (sterile
Environmental) throughout the entire time of the sterility test session.
The Letheen Agar plate is to be used to record the Operator's finger print impressions during
the test session or at its conclusion. If a needle stick injury occurs, before leaving the sterility
test room to change gloves, conduct a finger print impression. Use an additional Letheen
Agar plate to monitor the finger impressions at the conclusion of the session.
Four (4) contact plates are used for personnel monitoring of the hood, sleeve, chest and leg
after all testing has been completed, on exiting the change room.
NOTE: If a double consecutive session is being tested, perform personnel monitoring after
the second session only.
Record the Name, Date and Session details on the plates. Incubate all plates according to
MICLAB 045. Record results in the Monitoring Results for Sterility Test Sessions Finger,
Fallout, Environmental and Personnel Results File.
NOTE: After recording the finger print impression, the Operator must don a new pair of
sterile gloves in the change room and disinfect their hands with 70% IPA prior to resumption
of testing. See MICLAB 045 for limits.
3.3. Environmental Monitoring
Environmental monitoring must be performed with each Sterility Testing Session and in each
room and transfer hatch in use, on a daily basis. See the Table below for details:

Area Environmental Monitoring Required Frequency
Laminar Flow Cabinet 1000L Air Sample
1 x Contact Plate
Each Session
Each Session
Change Room 200L Air Sample
1 x Contact Plate (Floor)
Daily, when in use
Daily, when in use
Transfer Hatch 200L Air Sample
1 x Contact Plate
Daily, when in use
Daily, when in use
Sterility test Room 200L Air Sample
1 x Contact Plate (Floor)
1x Contact Plate (Trolley)
Daily, when in use
Daily, when in use
Daily, when in use
The environmental Agar plates and contact plates are to be labelled with all details and
incubated according to MICLAB 045. Results from the Laminar Flow monitoring are to be
entered in the Monitoring Results for Sterility Test Sessions Finger, Fallout, Environmental
and Personnel Results File. Results from ALL environmental samples are to be entered in
the Environmental Results Air / Surfaces Non-Sterile File . After the total count is recorded,
______________________________________________________________________________________

The product is to be regarded as a separate session with a known sterile control. No other
product is to be tested during this procedure.
a) Non-injectable, Non-filterable products
For non-injectable, non-filterable products follow the same procedure as for the
initial test. The known Sterile Control is Paraffin Oil in this test session.
b) Aqueous Solutions
1. For aqueous solutions the same quantity of product is filtered.
2. Include a known sterile control of X units of Water for Injection that has been
autoclaved at least twice in the test session.
3. Wash the canister filters with 3 x 100 mL of Sterile Peptone Water. These 3
washes should be individual. Fill the canisters with the appropriate medium,
seal and incubate as for an initial sterility test.
6.4. Recording of the session and results
Details of the test sessions and the results are to be recorded in the log book.
6.5. Interpretation of sterility repeat test results
6.5.1. If no evidence of microbial growth is found in the repeat test, the product examined
complies with the test for sterility. If microbial growth is found in the repeat test, the
product examined does not comply with the test for sterility.
7. Media Check after Sterility Testing (Stasis Test)
7.1. Membrane Filtration and Direct Inoculation
Once every 12 months media containers from a product tested by Membrane Filtration and
also if possible those from a product tested by Direct Inoculation must be subjected to a
challenge by a low number of microorganisms after the sterility test has been completed.
This is to ensure that any inhibitory effects present in the products have been successfully
inactivated by the dilution effect of the testing procedure, or the washing procedure as
appropriate to the Test Method, and hence any microorganisms that may have been present
in the product would have been able to grow in the media when the product was tested. It is
important that on an annual basis, the range of sterile products are to be subjected to a
Stasis Test.
A Stasis Check on Sterility Tests Form must be filled out on an annual basis detailing the
product type and date the Stasis test was conducted. NOTE: The product types listed are
not exhaustive; they represent the range of products manufactured by the company and list
only the specific products most likely to be inhibitory to the Sterility test.
7.2. See MICLAB 090 for the procedure of Stasis tests and to determine the method of
preparation of Stock Suspensions of micro organisms used for Stasis work and media
checks.
8. Validation and Revalidation of Sterility Test Methods
8.1. General Requirements
8.1.1 All products, which require sterility testing, must have a corresponding validated
method.
8.1.2 Validation is required for each new product formulation, changes in product
formulation, changes in containers / closure types and any other changes that
impact the status of a validated system, process or equipment.
8.1.3 Re-validations of existing methods are to be performed once every 5 years.
8.1.4 A revalidation of the sterility test system, using at least one product is to be
conducted on an annual basis.
______________________________________________________________________________________


9. Appendix 1 - Flowchart of Sterility Test results - Interpretation and Retests
Initial Test
Test
Result =
no growth
Test
Result =
Growth
Control
Result =
growth
Control
Result =
no growth
PASS
FAIL
Repeat test
Test result =
no growth
Test Result =
Growth
Problem with
session?
YES
NO
Does the
organism
match the
environment?
YES
NO

Form 680
Issue date:
Sterility Test Failure Investigation
(Ref. MICLAB 060)

Table Contents DR____________
1. Purpose of report
2. Batch Details
3. Batch Manufacturing review and Sterility testing
4. Summary of Conclusions found
5. Possible causes
6. Corrective action
7. Preventative action
8. Disposition of filling room
9. Documentation Approval of Investigation

1 Purpose of report
The purpose of this document is to provide a summary report of the incident investigation conducted into:

2 Batch Details
Product Name Batch #
Date of Manufacturing Filling Room #
Product Code Filling Machine
Container and size
Terminally sterilized Y/N Autoclave Cycle

Sterility Test Details
Date Technician
Test room
Number of products
tested

Time of test session
Session number that
day

Validated test method Tested to
Specification

Date of results Test session results
Contaminant/s type

Negative control
result

3 Batch review from manufacturing and testing.
Product Manufacturing Review
REVIEW AREA DETAILS & Results REVIEWED BY

Dispensing results:
Review of raw materials
used for the manufacture

Batch #
Materials code
Validated test method
Results of tests
Sign:
Date:
Review Attached: Yes/No
Dispensing of the raw
materials

Sign:
Date:
Sterilization cycles for
components
Autoclave
Cycle#
Date
Result
Sign:
Date:

Form 680
Issue date:
(Ref. MICLAB 060)
Interview of Operator for
any excursion of
procedures:
Name:

Date
Sign:
Date:
Training records
Gowning validations
Name
Training Record

Sign:
Date:
Operators Finger Dabs

Name
Results
Sign:
Date:
Operators Uniform

Name
Results

Sign:
Date:

Packing Line:
Autoclave cycle finished
product
Autoclave #
Cycle #
Result
Sign:
Date:
Issues on Inspection /
Finishing line

Sign:
Date:

QA Review Manufacturing:
Product Review of past
12 months of results

Sign:
Date:
Deviation Reports
Review

Sign:
Date:
Similar incidents

Sign:
Date:
Change control history
review

Sign:
Date:
Audit of security card
swipe

Sign:
Date:
Review of Media run
reports past 12 months

Sign:
Date:

Summary

Micro. Lab. to place a HOLD on adjacent & subsequent batches until establishing a cause which will
eliminate their risk of contamination and also;

Batches on HOLD (Q)
Batch # Code Product & % Container Size

Sterility Testing Review
REVIEW AREA DETAILS REVIEWED BY
Results of known sterile
controls

Sign:
Date:
Level of false positives in
routine testing (previous
12 months)

Sign:
Date:
Level false positives in
known sterile controls
previous 12 months)

Sign:
Date:
Review of sampling
procedures

Sign:
Date:
Date done: Notify QA Manager
Form 680
Issue date:
(Ref. MICLAB 060)
Interview of Technician

Sign:
Date:
Sterility Log Book

Sign:
Date:

Comparison of Isolates

Date of Streaking all Isolates:_________________
Technician:________________________________

Source of Isolate
No. of
Colonies

Colony Morphology
Microscopic
Appearance
Product

N/A

Finger Dab Plate

Uniform Plate

Laminar Flow Contact plate

Laminar Flow Air Sample

Fallout Plate

IPA Exp. Date:
Bucket 1

N/A

Bucket 2

N/A

4. Summary of Conclusions found

5. Possible cause/ Root Cause

MICLAB 060 Sample

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Standard Operating Procedure

Title: Micro Laboratory Procedure for Sterility Testing

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