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专业: 生物技术药物

[求助] 求助翻译一篇英文试验！！！事后重谢！！！

Tests for sterility and freedom from contamination
of biological products

Sophia Campbell
Bacteriology Section/Sterility Unit
Center for Veterinary Biologics – Policy, Evaluation, and Licensing
VS, APHIS, USDA
Ames, Iowa, USA

  A.  General procedures

Materials used in the production of biological products should be autoclaved
(sterilized with moist heat) and/or tested to ensure the absence of
contaminants before being used.  Samples of the finished biological product are
tested for bacterial, fungal, or mycoplasmal contaminants.

The assays for bacteria, Mycoplasma, and fungi described here are derived from
the assays required for veterinary biological products in the United States, as
regulated by the U.S. Department of Agriculture (Code of Federal Regulations,
Title 9, Subchapter E).
B.  Detection of bacteria and fungi

These assays describe materials and methods applicable for the detection of
bacteria and fungi by the direct inoculation of fluid media.

1.  General procedure for detecting viable bacteria and fungi

The Standard test for detecting extraneous bacteria and fungi in raw materials,
seed stocks, or final product is by direct inoculation of culture media.  A sterile
pipet or syringe with needle is used to aseptically transfer the biological
material directly into media.  If the biologic being tested has antimicrobial
properties, the ratio of the inoculum to the volume of culture media must be
determined before the test is started.  One hundred colony forming units (CFU)
of the control microorganisms listed in Table 1 are used to determine the
correct media volume to negate antimicrobial activity.  If the test sample
contains merthiolate as a preservative, fluid thioglycollate medium (FTM) is
used in test vessels incubated at both 30-350C and 20-250C.  If the test sample
is a killed biologic without merthiolate or a live bacterial biologic, FTM is used
at 30-350C and soybean casein digest medium (SCDM) at 20-250C.  If the test
sample is a live viral biologic, SCDM is used at both incubation temperatures.
If the inactivated bacterial vaccine is a clostridial biologic or contains a
clostridial component, the use of FTM with 0.5 percent added beef extract
(FTMB) in place of FTM is desirable.

Media for all sterility tests are incubated for not less than 14 days. At
intervals during incubation and on the 14th day of incubation, the test vessels
are examined for evidence of microbial growth.  Microbial growth can be
confirmed by subculture and Gram stain.

2.  Growth promotion and test interference

The sterility of the media should be confirmed by incubating representative
containers at the temperature and for the length of time specified for each test.

The ability of the culture media to support growth in the presence and absence
of product, product components, cells, seeds, or other test material should be
determined each time a test is performed.
To test for ability to support growth in the absence of the test material, media
should be inoculated with 10 to 100 viable control organisms of the suggested
American Type Culture Collection (ATCC) strains listed in Table 1 and
incubated according to the conditions specified. Ten tubes are checked for
each media and for both 10 and 100 viable control organisms. All tubes
inoculated with 100 organisms should show growth. Less than 10 tubes may
show growth with 10 control organisms.
TABLE 1
                                                                                    Incubation
Medium __Test Microorganism__________ Temperature (0C)__  Conditions
FTM          Bacillus subtilis-ATCC # 6633    30 - 35                Aerobic
FTM          Candida krusei-ATCC # 6258    20 - 25                Aerobic
SCDM    Bacillus subtilis-ATCC # 6633       30 - 35                Aerobic
SCDM    Candida krusei-ATCC # 6258       20 - 25                Aerobic
FTMB       Clostridium sporogenes-ATCC
                     # 11437                                     30 - 35                Anaerobic
To test for the ability of the culture media to support growth in the presence of
the test material, containers should be simultaneously inoculated with both
the test material and 100 viable control organisms.  Ten media containers
should be used to test each product.  The test media and volume are
satisfactory if clear evidence of growth of the control organisms appears in all
inoculated media containers within 7 days. The sterility test is considered
invalid if any of the media shows inadequate growth response of the organism
used to inoculate the material. Additional testing should be done on these
invalid products by increasing the volume of test media until there is clear
evidence of growth of the control organisms in 7 days.

3.  Number of items tested

Ten containers of all final product batches should be tested for sterility.  The
inoculum’s size per test container for live viral and bacterial products is 0.2
mL.  The inoculum’s size for killed viral and bacterial products is 1 mL per test
container. If a retest is conducted, then 20 containers of the final product
batch are tested.
4.  Interpretation of sterility test results

If growth is found in any media, but it can be demonstrated by controls that
the media or technique was faulty, then the first test is declared invalid and the
first test may be repeated.  If microbial growth is found in any of the test
vessels of the first test but there is no evidence invalidating it, then a retest
may be conducted.  The minimum number of biologic containers tested in a
retest is double the number of the first test.  If no growth is found in the first
test or retest, the biologic meets the requirements of the test and is considered
satisfactory for sterility.  If microbial growth is found in any of the retest
vessels, the biologic is considered unsatisfactory for sterility.  If, however, it can
be demonstrated by controls that the media or technique of the retest was
faulty, then the retest may be repeated.
  3
5.  General procedure for testing living viral vaccines produced in eggs (CEO)
and administered through drinking water, spray, or skin scarification for the
presence of bacteria and fungi

Each batch of final container biologic should have an average contamination of
not more than 1 bacterial or fungal colony per dose for vaccines recommended
for poultry or 10 colonies per dose for other animals.  Ten final container
product samples from each batch are tested.  From each container sample,
each of 2 petri plates are inoculated with vaccine equal to 10 doses, if the
vaccine is recommended for poultry, or 1 dose if recommended for other
animals.  Twenty mL of Brain Heart Infusion Agar containing 500 kinetic
Kersey units or 0.007 International units of penicillinase per mL should be

added to each of the plates.  One plate should be incubated at 30-350C for 7
days and the other at 20-250C.  Colony counts are made at the end of 14 days.
An average colony count of all the plates representing a batch shall be made for
each incubation condition.  If the average count at either incubation condition
exceeds 1 colony per dose for vaccines recommended for poultry or 10 colonies
per dose for vaccines recommended for other animals in the initial test, 1 retest
to rule out faulty technique may be conducted using 20 unopened final
containers.  If the average count at either incubation condition of the final test
for a batch exceeds 1 colony per dose for vaccines recommended for poultry or
10 colonies per dose for vaccines recommended for other animals, the batch of
vaccine should be considered unsatisfactory.

6.  General procedure for testing seed lots of bacteria and live bacterial
biologics for purity

Each seed lot of bacteria or batch of live bacterial biologic should be tested for
purity by inoculation of soybean-casein digest medium, which is incubated at
20-250C for 14 days, and fluid thioglycollate medium, which is incubated at
30-350C for 14 days.  Ten final container samples of each product batch are
tested. A sterile pipet or syringe and needle are used to aseptically transfer
0.2 mL of biologic directly into the two types of culture media.

If the inoculum or growth of the bacterial vaccine renders the medium turbid
so that the absence of atypical microbial growth cannot be determined by
visual examination, subcultures shall be made from all turbid tubes on the
14th day.  Subculturing is done by transferring 0.1 to 0.2 mL to differential
broths and agar and incubating for a 3 day period.  Microscopic examination
by Gram stain should also be done.
If no atypical growth is found in any of the test vessels when compared to a
positive control included with the test, the lot of biologic may be considered
satisfactory for purity.  If atypical growth is found but it can be demonstrated
by control that the media or technique was faulty, then the first test may be
repeated.  If atypical growth is found but there is no evidence invalidating the
test, then a retest may be conducted.  Twice the number of biologic containers
and test vessels of the first test are used in the retest.  If no atypical growth is
found in the retest, the biologic is considered satisfactory for purity.  If atypical
growth is found in any of the retest vessels, the biologic is considered
unsatisfactory for purity.  If, however, it can be demonstrated by controls that
the media or technique of the retest was faulty, then the retest may be
repeated.
  C.  Detection of Mycoplasma contamination

1.  General procedure for detecting Mycoplasma contamination

Each batch of live viral vaccine, each lot of Master Seed Virus (MSV), each lot of
primary and Master Cell Stock (MCS), and all ingredients of animal origin not
steam sterilized should be tested for absence of Mycoplasma.  Solid and liquid
Mycoplasma media, such as the formula specified in the 9CFR part 113.28,
that will support the growth of small numbers of test organisms including
Acholeplasma laidlawii (ATCC # 23206), Mycoplasma arginini (ATCC # 23838),
Mycoplasma hyorhinis (ATCC # 17981), and Mycoplasma orale (ATCC # 23714)
should be used.  For avian biologics, the test organism Mycoplasma synoviae
(ATCC # 25204) should also be used. Each batch of prepared Mycoplasma
broth and agar should be tested for growth promotion with at least one of these
test organisms.
One sample of each lot of vaccine, MSV, etc., should be tested.  Inoculate 1
plate of solid media with 0.1 mL of the sample being tested, and inoculate 100
mL of the liquid media with 1 mL of the sample.  Incubate the agar plate at 35-
370C aerobically in an atmosphere of air containing 5-10% CO2 and high
humidity for 10-14 days.  The Mycoplasma broth should also be incubated at
35-370C. On the 3rd or 4th day after inoculation, subculture from the liquid
media onto a plate of solid media with 0.1 mL.  Repeat the subculture
procedure on day 6 or 7, 9 or 10, and again on day 13 or 14.  Incubate all agar
plates for 10-14 days at 35-370C, aerobically in an atmosphere of air
containing 4-6% CO2 and high humidity.

2.  Interpretation of Mycoplasma test results

At the end of each plate's incubation period (10-14 days), examine the surface
of the solid media microscopically for the presence of Mycoplasma colonies.
The test sample passes the test if the growth of Mycoplasma colonies has not
occurred on any of the inoculated solid media.  If at any stage of the test more
than one plate is accidentally contaminated with bacteria or fungi or broken,
the test is invalid and should be repeated.  If Mycoplasma colonies are found on
any agar plate, the test should be repeated one time to confirm the Mycoplasma
contamination.  If Mycoplasma colonies are found on any of the agar plates of
the retest, the test sample should be considered unsatisfactory because of
Mycoplasma contamination.

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