.
1.0 Equipment Required
LAF
Manifold
holder assembly
Vacuum pump.
Forceps
Scissor
2.0 Material Required
Sample
Sterile
0.45µ Membrane filter
Sterile
FTM
Sterile
SCDM
Sterile
0.1% w/v Peptone water.
70%
sterile IPA Solution.
Gas
burner
Sterile
Filtration assembly
Sterile SCDA plates
Sterile Swab
3.0 Utilities Required
Vacuum Pump
4.0 Procedure:
4.1 Membrane Filtration Method
4.1.1 Sample
for Finished Products: Collect the samples to be tested for sterility as per SOP. Out of the whole
sample, randomly select 20 articles (as per Pharmacopoeia) of each lot of batch
for both LVP and SVP terminally sterilized products and for aseptic filled
products.
4.1.2
Sample for Intermediates: Randomly collect 16 pre-sterilized bottle samples to
be tested for sterility from LVP bottle pack machine (When change over the new
Product collect the 16 bottle samples of Ist batch ) separately in
such a way that each bottle should represent each cavity of the mould of bottle
pack machine.
4.1.3
Transfer the samples to QC department in clean plastic crates.
4.1.4
Wipe the sample article individually with 70% IPA solution and keep in a clean
S.S trays marked with Product Name, Batch No and Lot No, and then transfer the
samples to the sterility room through clean pass box for performing sterility.
4.1.5
Prepare the media tubes (FTM and SCDM) as per the SOP for preparation of culture media, dispense 100-100 ml quantity in test tubes & plug them.
Sterilize both the media at 121ºC and 15 psi pressure for 20 minutes as per SOP for Media Sterilization by Autoclaving.
4.1.6
After autoclaving Label the tubes with Name of Media, Media Batch No. and
pre-incubate the media tubes at appropriate temperature i.e. SCDM tubes at 20
to 25ºC whereas FTGM tubes at 30 to 35ºC for 24 - 48 hrs before subjecting them
for sterility operations.
4.1.7
Autoclave Dress, S.S. cups, Receptacle unit, Scissors and forceps in a stainless steel container at 121ºC temperature and 15psi pressure for 30 minutes as per SOP.
4.1.8
After Sterilization cool the contents and aseptically transfer in a S.S.
container to cleaned Pass box.
5.4.1.9
Transfer the pre incubated sterile media tubes, SCDA plates, sterile swabs,
sterilized manifold, and 0.1% w/v peptone water to the sterility test room
through pass box.
4.1.10 Enter
in sterility room as per the SOP for Entry / Exit procedure for Sterility Room.
4.1.11 Start
the LAF as per SOP for operating instruction for LAF.
4.1.12 Wipe
out all samples to be tested for sterility with 70% IPA solution.
4.1.13 Before
starting sterility test, expose the SCDA plates as specified locations
throughout the testing as per SOP.
4.1.14 Connect
the Filtration manifold holder assembly with the S.S. reservoir properly with
pipe and place sterilized S.S. cups in the sterile receptacle under Laminar airflow unit. Check the Manometer reading of working LAF and check the
temperature as well as humidity of the sterility room
4.1.15 Manometer
reading of working LAF chamber pressure should be between 08 – 15mm of WG. Temperature
reading of the sterility room should be 27ºC ± 2ºC.
4.1.16 Switch
ON the vacuum pump but close the vacuum of the manifold holder assembly with
the help of vacuum control key, present on the base of individual manifold
holder. Now place 0.45m sterile membrane filters between filtration cup and
receptacle with the help of sterilized forceps.
4.1.17 Wet
the membrane filter by adding approx 15 ml of sterilized Fluid A (0.1% peptone
water) to filter holder, and filter the fluid by employing vacuum.
4.1.18 Cut
the tip of bottle/vial or ampoule with sterile SS blade in front of the gas
burner and immediately transfer not less than half of the contents for LVP and
whole content of the vial for SVP to membrane.
4.1.19 Immediately
filter the solution with the aid of vacuum and wash the membrane three times
with 100 ml of sterilized fluid A (Washing with fluid A is not required in case
of sterile water for injection).
4.1.20 After
complete filtration, stop the vacuum of manifold with the help of manifold
vacuum control key.
4.1.21 Lift
the membrane carefully with the help of sterile forceps, aseptically cut the
membrane filter into two halves with sterile SS scissor and transfer one half
to FTM and one half to SCDM tubes by unplugging in front of gas burner only.
4.1.22 Label
both the tubes with product name, B. No, lot No., date of testing, Completion
date & Tested by.
4.1.23 Simultaneously
prepare a negative control by filtering 100 ml of 0.1% peptone water instead of
product sample, cut the membrane into two halves with sterile SS scissor and
transfer one half to FTM and one half to SCDM and label both the tubes as
Negative control.
4.1.24 Simultaneously
prepare a chamber control during the sterility take two tubes, one is SCDM
& other one is FTM tube, unplug the cotton plug of the tube and expose in
LAF during sterility, after completion of sterility re-plug the tubes and then
incubate the tubes as a chamber control.
4.1.25 After
completion of work transfer all inoculated media through hatch box and then
transfers all the equipment and exposed plates to microbiology analysis
section.
4.1.26 Immediately
move from the sterility area as per exit procedure specified SOP for
Entry Exit Procedure for Sterility Room.
4.1.27 Incubate
the FTM tubes at 30ºC – 35ºC and SCDM tubes at 20ºC – 25ºC for 14 days.
Incubation
period for terminally sterilized products is not less than 7 days and 14 days
for aseptically filled products as per IP. If the Product is as per USP, BP,
incubation period is 14 days for both terminally sterilized as well as for
aseptically filled products.
4.1.28 Start
the LAF of Microbiology Analysis room as per SOP and prepare four positive
Control tubes by inoculating aseptically 10 to 100 cfu in FTM tubes with S.
aureus NCIM 2079, P. aeruginosa NCIM 2200, B.
subtilis NCIM 2063 and environmental flora EF - 1. Similarly
prepare three SCDM positive control by inoculating approx 10 to 100 cells
separately with C. albicans NCIM 3471, A. niger NCIM
1196 and environmental flora EF - 2. Incubate FTM positive control tubes at 30
– 35ºC for 3 days & SCDM positive control tubes at 20 – 25ºC for 5 days.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria
Related: Incubation Conditions for Common Media used for Fungus and Bacteria
4.2 Observation and Interpretation of Results
4.2.1
Visually examine the media tubes daily to its conclusion for macroscopic
evidence of microbial growth.
4.2.2
If no evidence of growth observed in any of the tube the product to be examined
for the test complies with the test for sterility.
4.2.3
The test is not valid unless the negative control shows negative till at end of
incubation, and positive control shows growth within specified incubation
period.
4.2.4
Destroy all the positive controls after confirmation of growth in both FTM and
SCDM tubes (Generally growth will observed in 24 to 48 hrs).
4.2.5
If evidence of microbial growth is found in any of the tube, investigate the
cause of its failure as per SOP for sterility test failure investigation.
If investigation report concludes that test is invalid, repeat the test with 20
units.
4.2.6
If no evidence of growth is found in the repeat test the product examined
complies with the test for sterility. If evidence of microbial growth is found
in the repeat test the product examined does not comply with the test for
sterility.
Related: Incubation of Petridishes in Inverted Position
Related: Incubation of Petridishes in Inverted Position
5.0 Precaution
5.1
Sample bottles / vials should be wiped properly with filtered 70% IPA before
transferring them to sterility room and also inside the sterility room before
sterility operations. Hatch box should also be cleaned properly with filtered
70% IPA solution.
5.2
Any material used in the sterility operation or inside sterility room should be
autoclaved properly.
5.3
The tip of sample bottles / vials should be heated properly in front of gas
burner before cutting with sterile heated cutting blade.
5.4
Clean the waste solution reservoir immediately after sterility operations and
mop the outer surface of the reservoir with filtered 70% IPA with sterile
mopper.
6.0 Frequencies
For LVP – Lot wise
For SVP – Batch wise
7.0 Abbreviation
SOP
: Standard Operating Procedure
IPA : Isopropyl Alcohol
SVP : Small volume
Parenterals
LVP : Large
volume Parenterals
FTM : Fluid
Thioglycollate Medium
SCDM : Soybean casein digest
medium
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