[Title 21 CFR 630]
[Code of Federal Regulations (annual edition) - April 1, 1996 Edition]
[Title 21 - FOOD AND DRUGS]
[Chapter I - FOOD AND DRUG ADMINISTRATION,]
[Subchapter F - BIOLOGICS]
[Part 630 - ADDITIONAL STANDARDS FOR VIRAL VACCINES]
[From the U.S. Government Publishing Office]
21
FOOD AND DRUGS
7
1996-04-01
1996-04-01
false
ADDITIONAL STANDARDS FOR VIRAL VACCINES
630
PART 630
FOOD AND DRUGS
FOOD AND DRUG ADMINISTRATION,
BIOLOGICS
PART 630--ADDITIONAL STANDARDS FOR VIRAL VACCINES--Table of Contents
Subpart A--Poliovirus Vaccine Inactivated
Sec.
630.1 Poliovirus Vaccine Inactivated.
630.2 Poliovirus Vaccine Inactivated.
630.3 Potency test.
630.4 Tests for safety.
630.5 General requirements.
Subpart B--Poliovirus Vaccine Live Oral Trivalent
630.10 Poliovirus Vaccine Live Oral Trivalent.
630.11 Clinical trails to qualify for license.
630.12 Animal source and quarantine; personnel.
630.13 Manufacture of Poliovirus Vaccine Live Oral Trivalent.
630.14 Reference virus preparations.
630.15 Potency test.
630.16 Test for neurovirulence.
630.17 Alternative test for neurovirulence.
630.18 Additional tests for safety.
630.19 General requirements.
Subpart C--[Reserved]
Subpart D--Measles Virus Vaccine Live
630.30 Measles Virus Vaccine Live.
630.31 Clinical trials to qualify for license.
630.32 Manufacture of live, attenuated Measles Virus Vaccine.
630.33 Reference virus.
630.34 Potency test.
630.35 Test for safety.
630.36 General requirements.
Subpart E--[Reserved]
Subpart F--Mumps Virus Vaccine Live
630.50 Mumps Virus Vaccine Live.
630.51 Clinical trials to qualify for license.
630.52 Manufacture of Mumps Virus Vaccine Live.
630.53 Reference virus.
630.54 Potency test.
630.55 Test for safety.
630.56 General requirements.
Subpart G--Rubella Virus Vaccine Live
630.60 Rubella Virus Vaccine Live.
630.61 Clinical trials to qualify for license.
630.62 Production.
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630.63 Reference virus.
630.64 Potency test.
630.65 Test for safety.
630.66 General requirements.
Subpart H--Smallpox Vaccine
630.70 Smallpox Vaccine.
630.71 Production.
630.72 Reference vaccine.
630.73 Potency test.
630.74 Tests for safety.
630.75 General requirements.
Authority: Secs. 201, 501, 502, 503, 505, 510, 701 of the Federal
Food, Drug, and Cosmetic Act (21 U.S.C. 321, 351, 352, 353, 355, 360,
371); secs. 215, 351, 352, 353, 361 of the Public Health Service Act (42
U.S.C. 216, 262, 263, 263a, 264).
Source: 38 FR 32068, Nov. 20, 1973, unless otherwise noted.
Cross References: For U.S. Customs Service regulations relating to
viruses, serums, and toxins, see 19 CFR 12.21--12.23. For U.S. Postal
Service regulations relating to the admissibility to the United States
mails see parts 124 and 125 of the Domestic Mail Manual, that is
incorporated by reference in 39 CFR part 111.
Subpart A--Poliovirus Vaccine Inactivated
Sec. 630.1 Poliovirus Vaccine Inactivated.
(a) Proper name and definition. The proper name of this product
shall be ``Poliovirus Vaccine Inactivated'' which shall consist of an
aqueous preparation of poliovirus types 1, 2, and 3, grown in monkey
kidney tissue cultures, inactivated by a suitable method.
(b) Strains of virus. Strains of poliovirus used in the manufacture
of vaccine shall be identified by historical records, infectivity tests
and immunological methods. Any strain of virus may be used that produces
a vaccine meeting the requirements of Secs. 630.2, 630.3, and 630.4, but
the Director, Center for Biologics Evaluation and Research may from time
to time prohibit the use of any specific strain whenever he finds that
it is practicable to use another strain of the same type that is
potentially less pathogenic to man and that will produce a vaccine of at
least equivalent safety and potency.
(c) Monkeys; species permissible as source of kidney tissue. Only
Macaca or Cercopithecus monkeys, or a species found by the Director,
Center for Biologics Evaluation and Research, to be equally suitable,
which have met all requirements of Secs. 600.11(f)(2) and 600.11(f)(8)
of this chapter shall be used as a source of kidney tissue for the
manufacture of Poliovirus Vaccine Inactivated.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 50
FR 4137, Jan. 29, 1985; 55 FR 11013, Mar. 26, 1990]
Sec. 630.2 Poliovirus Vaccine Inactivated.
(a) Cultivation of virus. Virus for manufacturing vaccine shall be
grown with aseptic techniques in monkey kidney cell cultures. Suitable
antibiotics in the minimum concentration required may be used
(Sec. 610.15(c) of this chapter).
(b) Filtration. Within 72 hours preceding the beginning of
inactivation, the virus suspensions shall be filtered or clarified by a
method having an efficiency equivalent to that of filtration through an
S1 Seitz type filter pad.
(c) Virus titer. The 50 percent endpoint (TCID50) of the virus
fluids after filtration shall be 106.5 or greater as confirmed by
comparison in a simultaneous test (using groups of 10 tubes at 1 log
steps or groups of 5 tubes at 0.5 log steps) with a reference virus
distributed by the Center for Biologics Evaluation and Research.
Acceptable titrations of the reference virus shall not vary more than
plus-minus0.5 log10 from its labeled titer using 0.5
milliliter inoculum in tissue culture.
(d) Inactivation of virus. The virus shall be inactivated, as
evidenced by the tests described in Sec. 630.4, through the use of an
agent or method which has been demonstrated to be consistently effective
in the hands of the manufacturer in inactivating a series of lots of
poliovirus. If formaldehyde is used for inactivation, it shall be added
to the virus suspension to a final concentration of U.S.P. solution of
formaldehyde of 1:4000, and the inactivation conducted under controlled
conditions of pH and time, at a temperature of 36 deg. to 38 deg. C.
Three or more virus titers, suitably spaced to indicate rate of
inactivation, shall be determined during the inactivation process.
Filtration equivalent to that described in paragraph (b) of this section
shall be performed after the estimated baseline time (time at which the
50 percent end-
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point reaches one tissue culture infective dose per milliliter), but
prior to sampling for the first single strain tissue culture test
required in Sec. 630.4(b), except that this filtration may be omitted
for strains of a virulence for monkeys equal to or less than that of the
MEF-1 Type 2 strain of poliovirus.
(e) Additional processing. Single strain or trivalent pools that
have failed to pass safety tests prescribed in Sec. 630.4 (b), (c), or
(e) may be treated as follows:
(1) Filtration or clarification by a method having an efficiency
equivalent to that of filtration through an S1 Seitz type filter pad.
(2) Negative tests performed as described in Sec. 630.4 (b) and (c)
must be obtained on each of two successive samples taken so as to be
separated by an interval of at least 3 days while the material is being
subjected to treatment with 1:4000 U.S.P. formaldehyde solution and heat
at 36 deg. to 38 deg. C. The first sample may be taken before incubation
is begun and the second sample shall be taken after the incubation of at
least 3 days is completed. For both single strain and trivalent pools
the volume tested for each tissue culture safety test shall be
equivalent to at least 1,500 human doses.
(3) Pools which are positive following such additional processing
shall not be used for the manufacture of Poliovirus Vaccine Inactivated
(f) Supplemental inactivation. Supplemental inactivation employing a
method capable of reducing the titer of a similarly produced virus
suspension by a factor of 10-6 may be applied at any point after
the filtration step described in paragraph (d) or (e)(1) of this
section.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 50
FR 4137, Jan. 29, 1985; 55 FR 11013, Mar. 26, 1990]
Sec. 630.3 Potency test.
Each lot of vaccine shall be subjected to a potency test which
permits an estimation of the antigenic capacity of the vaccine. This is
done by means of a simultaneous comparison of the serum antibody levels
produced in monkeys by the vaccine under test with the antibody level of
the reference serum distributed by the Center for Biologics Evaluation
and Research. The potency test shall be performed on samples taken after
all final processing of the product has been completed, including
addition of preservative, except that when the final product contains
material having an adjuvant effect an additional test shall be performed
with a sample taken before the addition of the adjuvant material. The
volume of the test sample for the additional test shall be adjusted to
the equivalent volume of Poliovirus Vaccine Inactivated in the final
product. The test shall be conducted as follows:
(a) Inoculation of monkeys. A group of 12 or more Macaca monkeys, or
a species found by the Director, Center for Biologics Evaluation and
Research, to be equally suitable for the purpose, shall be used. Animals
shall weigh between 4 and 8 pounds and shall be in overt good health.
Animals that become ill and remain ill during the course of immunization
shall be excluded from the group. The test shall not be valid unless at
least 10 animals survive the test period and their preinoculation serum
antibody levels are as prescribed in paragraph (d) of this section. The
test vaccine shall be given intramuscularly to each monkey in 3 doses at
7-day intervals, each dose to be the recommended individual human dose.
Only undiluted vaccine shall be used.
(b) Serum samples. A blood sample shall be taken from each monkey
prior to vaccination and then again 7 days after the last injection.
Serum shall be separated aseptically, and stored under refrigeration.
(c) Serum-virus neutralization test. The titers of individual monkey
serums shall be determined in comparison with the reference serum in
tests designed to include controls for all the variables of significance
including the following:
(1) Serum toxicity control;
(2) Cell control and cell titration;
(3) Virus titration control (at least 4 tubes for each dilution at
0.5 log steps); and
(4) Serum controls using type-specific serums to identify the type
of virus used in the neutralization test.
(d) Interpretation of the test. Animals showing preinoculation
titers of 1:4 or over when tested against not more
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than 1,000 TCID50 of virus, shall be excluded from the test. The
geometric mean titer of antibody induced in the monkeys surviving the
course of immunization and bleeding, shall be calculated. A comparison
of the value so obtained shall be made with the value for the reference
serum that was tested simultaneously and expressed as the ratio between
the geometric mean titer value of the serums under test and the mean
titer value of the reference serum.
(e) Potency requirements. A lot of vaccine tested against the
reference serum shall be satisfactory if the geometric mean value of the
group of individual monkey serums representing the lot of vaccine tested
is at least 1.29 times the mean value of the reference serum for Type 1,
at least 1.13 times for Type 2, and at least 0.72 times for Type 3.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 50
FR 4137, Jan. 29, 1985; 55 FR 11013, Mar. 26, 1990]
Sec. 630.4 Tests for safety.
In the manufacture of the product, the following tests relating to
safety shall be conducted by the manufacturer.
(a) The virus pool--tests prior to inactivation--(1) B virus and
Mycobacterium tuberculosis. Prior to inactivation, each individual virus
harvest or virus pool shall be tested for the presence of B virus and
Mycobacterium tuberculosis.
(2) SV-40. Prior to inactivation, the material shall be tested for
the presence of SV-40 as follows (or by any other test producing equally
reliable results): A sample of at least 5 ml. from the virus harvest or
virus pool shall be neutralized by high titer specific antiserum of
other than primate origin. A similar sample from the pool of tissue
culture fluids from control vessels representing the tissue from which
the virus was prepared may be tested in place of the virus sample. The
sample shall be tested in primary cercopithecus tissue cultures or in a
cell line demonstrated as at least equally susceptible to SV-40. Each
tissue culture system shall be observed for at least 14 days and at the
end of the observation period at least one subculture of fluid shall be
made in the same tissue culture system and the subculture shall be
observed for at least 14 days.
(3) Test results. The virus harvest or virus pool is satisfactory
for poliovirus vaccine only if the tests produce no evidence of the
presence of B virus, Mycobacterium tuberculosis or SV-40.
(b) Single strain pool tissue culture tests for poliovirus. (1)
Before pooling to make the final poliovirus vaccine, during inactivation
at 36 deg. to 38 deg. C., two samples of each monovalent bulk strain
pool shall be tested for the presence of virus by tissue culture
methods, the second sample to be taken at least 3 days after taking the
first sample.
(2) Each sample shall be no smaller than the equivalent of 1,500
human doses and shall be subjected to the complete testing process and
each test shall be performed on a different monkey kidney tissue culture
cell preparation. The test sample for one of these tests may be used
also for the test prescribed in paragraph (f) of this section provided
the cell cultures used have been demonstrated as fully susceptible to
SV-40 and poliovirus. Each sample shall be inoculated into five or more
tissue culture bottles of a suitable capacity, the ratio of the vaccine
to the nutrient fluid being approximately 1:1 to 1:3, and the area of
the surface growth of cells being at least 3 square centimeters per
milliliter of sample. The tissue culture bottles shall be observed for
at least 14 days.
(3) A first subculture shall be made at the end of 7 days from date
of inoculation by planting at least 2 percent of the volume from each
original bottle into suitable tissue culture vessels, followed by
refeeding.
(4) A second subculture shall be made from each original bottle in
the same manner at the end of 14 days from date of inoculation.
(5) Each of the first and second subcultures shall be observed for
at least 7 days.
(6) If cytopathogenic effects occur either in the original bottles
of the two tests or in the subcultures from them, or if cellular
degeneration appears in the original bottles or in the subcultures
before degeneration occurs in uninoculated cultures, the pool shall be
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held until the matter is resolved. If active poliovirus is indicated,
the strain pool shall not be used for inclusion in a final vaccine
unless effectively reprocessed as described in Sec. 630.2(e). If other
viruses are present, the pool shall not be used unless it can be
demonstrated that such viruses have originated from other than the
strain pool being tested.
(c) Trivalent vaccine pool tissue culture test. No less than 1,500
human doses of the trivalent vaccine pool, without final preservative,
prepared by pooling the three type pools, each of which has passed all
tests prescribed in paragraph (b) of this section, shall be subjected to
the complete tissue culture test prescribed in such paragraph (b) in at
least two approximately equal tests in separate monkey kidney tissue
culture preparations. This test sample may be used also for the test
prescribed in paragraph (f) of this section provided the cell cultures
used have been demonstrated as fully susceptible to SV-40 and
poliovirus.
(d) Trivalent vaccine pool lymphocytic choriomeningitis test. The
final vaccine shall be shown to be free of lymphocytic choriomeningitis
virus by intracerebral inoculation of the maximum volume tolerated into
10 or more mice which shall be observed daily for at least 21 days and a
negative test shall not be valid unless at least eight mice survive for
this period.
(e) Test in monkeys for active virus. (1) Vaccine from final
containers selected at random from each filling of each lot shall be
pooled to provide a test sample of at least 400 milliliters representing
the various fillings. An equal volume of bulk vaccine may be substituted
for test samples from each filling lot provided the procedure has been
approved by the Director, Center for Biologics Evaluation and Research.
(2) A total of not less than 20 monkeys shall be inoculated with the
test sample. A preinjection serum sample from each monkey must not
contain neutralizing antibody against the three poliovirus types
detectable in a dilution of 1:4 when tested against not more than 1,000
TCID50 of virus. At least 80 percent of the test animals
representing each filling or each bulk sample must survive the test
period without significant weight loss, except that if at least 60
percent of the test animals survive the first 48 hours after injection,
those animals which do not survive this 48-hour test period may be
replaced by an equal number of test animals. At least 80 percent of the
animals used in the test must show microscopic evidence of inoculation
trauma in the lumbar region of the spinal cord, and gross or microscopic
evidence of inoculation trauma in the thalamic area. If less than 60
percent of the test animals survive the first 48 hours, or if less than
80 percent of the animals fail to meet the other criteria prescribed in
this section, the test must be repeated.
(3) Vaccines shall be injected by combined intracerebral,
intraspinal, and intramuscular routes into Macaca or Cercopithecus
monkeys or a species found by the Director, Center for Biologics
Evaluation and Research, to be equally suitable for the purpose. The
animals shall be in overt good health and injected under deep
barbiturate anesthesia. The intracerebral injection shall consist of 0.5
milliliter of test sample into the thalamic region of each hemisphere.
The intraspinal injection shall consist of 0.5 milliliter of
concentrated test sample into the lumbar spinal cord enlargement, the
test sample to be concentrated 100 fold in the ultracentrifuge by a
method demonstrated to recover at least 90 percent of the virus
particles in the sediment after it has been resuspended in the same lot
of unconcentrated test sample. The intramuscular injection shall consist
of 1.0 milliliter of test sample into the right leg muscles. At the same
time, 200 milligrams of cortisone acetate shall be injected into the
left leg muscles, and 1.0 milliliter of procaine penicillin (300,000
units) into the right arm muscles. The monkeys shall be observed for 17
to 19 days and signs suggestive of poliomyelitis shall be recorded.
(4) At the end of the observation period, samples of cerebral cortex
and of cervical and lumbar spinal cord enlargements shall be taken for
virus recovery and identification. Histological sections shall be
prepared from both spinal cord enlargements and examined.
[[Page 90]]
(5) Doubtful histopathological findings necessitate (i) examination
of a sample of sections from several regions of the brain in question,
and (ii) attempts at virus recovery from the nervous tissues previously
removed from the animal. The test results must be negative. Test results
are negative if the histological and other studies leave no doubt that
poliovirus infection did not occur.
(f) Tissue culture safety test for SV-40. At least 500 human doses
of each monovalent or trivalent pool of vaccine shall be tested for the
presence of SV-40 using primary cercopithecus monkey tissue cultures or
using a cell line demonstrated as at least equally susceptible to SV-40.
The test shall be conducted as described in paragraph (b) of this
section, except for the volume of test sample and except that one
subculture of at least 2 percent of the volume of the fluids shall be
made no less than 14 days from the date of inoculation and examined for
at least 14 days from the date of subinoculation. The vaccine is
satisfactory only if there is no evidence of the presence of SV-40 in
any of the cultures or subcultures.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 50
FR 4137, Jan. 29, 1985; 50 FR 16229, Apr. 25, 1985; 55 FR 11013, Mar.
26, 1990; 57 FR 10814, Mar. 31, 1992]
Sec. 630.5 General requirements.
(a) Consistency of manufacture. No lot of final vaccine shall be
released unless it is one of a series of five consecutive lots produced
by the same manufacturing process, all of which have shown negative
results with respect to all tests for the presence of live poliovirus,
and unless each of the monovalent pools of which a polyvalent final
vaccine is composed similarly is one of a series of five consecutive
monovalent pools of the same type of inactivated poliovirus, all of
which have shown negative results in all tests for the presence of live
poliovirus.
(b) Dose. These additional standards are based on a human dose of
1.0 milliliter for a single injection and a total human immunizing dose
of three injections of 1.0 milliliter given at appropriate intervals.
(c) Samples and protocols. For each lot of vaccine, the following
material shall be submitted to the Director, Center for Biologics
Evaluation and Research, Food and Drug Administration, 8800 Rockville
Pike, Bethesda, MD 20892:
(1) A 2,500 milliliter sample, neutralized, not dialyzed, and
without final preservative, taken at the latest possible stage of
manufacturing before the addition of such preservative.
(2) A 200 milliliter bulk sample of the final vaccine containing
final preservative.
(3) A total of not less than a 200 milliliter sample of the final
vaccine in final labeled containers.
(4) A protocol which consists of a summary of the history of
manufacture of each lot including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 51
FR 18580, May 21, 1986; 55 FR 11013, Mar. 26, 1990]
Subpart B--Poliovirus Vaccine Live Oral Trivalent
Source: 56 FR 21432, May 8, 1991, unless otherwise noted.
Sec. 630.10 Poliovirus Vaccine Live Oral Trivalent.
(a) Proper name and definition. The proper name of this product
shall be Poliovirus Vaccine Live Oral Trivalent. The vaccine shall be a
preparation containing the three types of live, attenuated polioviruses
grown in monkey kidney cell cultures, or in a cell line found by the
Director, Center for Biologics Evaluation and Research, to meet the
requirements of Sec. 610.18(c) of this chapter. The vaccine shall be
prepared in a form suitable for oral administration.
(b) Criteria for acceptable strains. (1) The Sabin strains of
attenuated poliovirus, Type 1 (LS-c, 2ab/KP2), Type 2 (P712, Ch,
2ab/KP2), and Type 3 (Leon 12a1b/KP3), or derivatives
from them, may be used in the manufacture of vaccine.
(2)(i) Other poliovirus strains may be used in the manufacture of
Poliovirus Vaccine Live Oral Trivalent provided that they are identified
by historical records including:
(A) Origin,
[[Page 91]]
(B) Techniques of attenuation,
(C) Antigenic properties,
(D) Neurovirulence for monkeys,
(E) Pathogenicity for tissue cultures of various cell types, and
(F) Established virus markers, including rct/40, and d.
(ii) The data shall be submitted to the Director, Center for
Biologics Evaluation and Research, along with other data that establish:
(A) That each such strain is at least as safe as the Sabin strain of
the corresponding type,
(B) That each such strain demonstrates results comparable to the
Sabin srain when inoculated into monkeys by the intrathalamic and
intramuscular routes, and
(C) That each such strain has been used to produce vaccines meeting
the safety and potency requirements of Secs. 630.11, 630.15, 630.16 or
630.17, and 630.18.
(3) The Director, Center for Biologics Evaluation and Research, may
prohibit the use of a specified strain whenever the Director finds that
it is practicable to use another strain of the same type that will
produce a vaccine of greater safety and of at least equivalent potency.
(4) If vaccine lots have been produced directly from strain
materials (e.g., Sabin Original, Sabin Original Merck, or Sabin Original
Rederived), the strain material is not required to be tested in
accordance with the provisions of Sec. 630.10(c).
(c) Criteria for qualification of the seed virus. (1) Each seed
virus used in vaccine manufacture shall be prepared from an acceptable
strain in monkey kidney cell cultures, derived from animals which have
met all of the requirements of Sec. 630.12(a), or in a cell culture of a
type determined to be suitable by the Director, Center for Biologics
Evaluation and Research. The seed virus used in vaccine manufactures
shall be demonstrated to be free of extraneous microbial agents except
for unavoidable bacteriophage.
(2) Seed virus used for the manufacture of oral poliovirus vaccine
shall meet the requirements of Secs. 630.13, 630.16 or 630.17, and
630.18. In addition, the neurovirulence of each of the first five
consecutive monovalent virus pools prepared from the seed virus shall
meet the neurovirulence requirements prescribed in Secs. 630.16(b)(2) or
630.17 (b)(3).
(3) A new seed virus may be used for production provided data are
submitted in the form of a product license a supplement that show the
new seed virus and each of the first five consecutive monovalent virus
pools prepared from it meet the safety requirements of Secs. 630.13 and
630.16 or 630.17 and 630.18 and approval for the use of the seed virus
is received in writing from the Director, Center for Biologics
Evaluation and Research.
(4) Seed virus in vaccine manufacture shall be prepared in a seed
lot system from a master virus seed lot at a passage level consistent
with Sec. 630.13(a).
(5) For monovalent virus pools tested in accordance with
Sec. 630.16(b), the use of the seed virus may continue provided that the
frequency of monovalent virus pools produced with it which fail to meet
the criteria of neurovirulence for monkeys prescribed in
Sec. 630.16(b)(2) is not greater than predicted on the basis of
comparison with the corresponding reference preparation. If the
frequency of monovalent virus pools produced with the same seed virus
which fail to meet the criteria of neurovirulence for monkeys prescribed
in Secs. 630.16(b)(2) is greater than the predicted 1 percent on the
basis of the 99-percent fiduciary one-sided upper limit, that seed virus
shall be disqualified for further use in vaccine production.
(6) For monovalent virus pools tested in accordance with
Sec. 630.17, subsequent and identical neurovirulence tests of the seed
virus shall be performed in monkeys whenever there is evidence of a
significant increase in the neurovirulence of the seed virus, upon
introduction of a new production seed lot, and as often as is necessary
to otherwise establish, to the satisfaction of the Director, Center for
Biologics Evaluation and Research, that the seed virus for vaccine
manufacture has maintained its neurovirulence properties as set forth in
Sec. 630.17 (b)(3).
[56 FR 21432, May 8, 1991, as amended at 59 FR 49351, Sept. 28, 1994]
[[Page 92]]
Sec. 630.11 Clinical trials to qualify for license.
To qualify for license, the antigenicity of the vaccine shall have
been determined by clinical trials of adequate statistical design
conducted in compliance with part 56 of this chapter, unless exempted
under Sec. 56.104 or granted a waiver under Sec. 56.105, and with part
50 of this chapter. Such clinical trials shall be conducted with five
lots of oral poliovirus vaccine that have been manufactured by the same
methods. Type specific neutralizing antibody for each type of poliovirus
in the vaccine shall be induced in 90 percent or more of susceptibles
after a series of doses.
Sec. 630.12 Animal source and quarantine; personnel.
(a) Monkeys--(1) Species permissible as source of kidney tissue.
Only Macaca monkeys, Cercopithecus monkeys, or other species found by
the Director, Center for Biologics Evaluation and Research, to be
equally suitable, which meet the requirements of Sec. 600.11 (f)(2) and
(f)(8) of this chapter, shall be used as the source of kidney tissue for
the manufacture of Poliovirus Vaccine Live Oral Trivalent.
(2) Experimental and test monkeys. Monkeys that have been used
previously for experimental or test purposes shall not be used as a
source of kidney tissue in the processing of vaccine.
(3) Quarantine; additional requirements. Excluding deaths from
accidents or causes not due to infectious diseases, if the death rate of
any group of monkeys being conditioned in accordance with
Sec. 600.11(f)(2) of this chapter exceeds 5 percent per month, the
remaining monkeys may be used for the manufacture of Poliovirus Vaccine
Live Oral Trivalent only if all of the monkeys survive a new quarantine
period.
(b) Personnel. All reasonably possible steps shall be taken to
ensure that personnel involved in processing the vaccine are immune to
all three types of poliovirus and do not excrete poliovirus.
[56 FR 21432, May 8, 1991; 56 FR 27787, June 17, 1991]
Sec. 630.13 Manufacture of Poliovirus Vaccine Live Oral Trivalent.
(a) Virus passages. Virus in the final vaccine shall represent no
more than five tissue culture passages from the original strain or no
more than five tissue culture passages from a virus clone derived from
one of the first five tissue culture passages of the original strain.
(b) Virus propagated in primary monkey kidney cell cultures--(1)
Continuous cell lines. When primary monkeys kidney cell cultures are
used in the manufacture of poliovirus vaccine, continuous cell lines
shall not be introduced or propagated in vaccine manufacturing areas.
(2) Identification of processed kidneys. The kidneys from each
monkey shall be processed separately. The resulting viral fluid shall be
identified as a separate monovalent harvest and kept separately from
other monovalent harvests until all samples for the tests prescribed in
paragraphs (b)(3) and (b)(4) of this section relating to that pair of
kidneys have been withdrawn from the harvest.
(3) Monkey kidney tissue production vessels prior to virus
inoculation. Prior to inoculation with the seed virus and at least 3
days after complete formation of the tissue sheet, the tissue culture
growth in vessels derived from each pair of kidneys shall be examined
microscopically for evidence of cell degeneration. If such evidence is
observed, the tissue cultures from that pair of kidneys shall not be
used for poliovirus vaccine manufacture. To test the tissue found free
of cell degeneration for further evidence of freedom from demonstrable
viable microbial agents, the fluid shall be removed from the cell
cultures immediately prior to virus inoculation and tested in each of
four culture systems:
(i) Macaca monkey kidney cells,
(ii) Cercopithecus monkey kidney cells,
(iii) Primary rabbit kidney cells, and
(iv) Cells from one of the systems described in Sec. 630.18(a)(6).
The fluid shall be tested in the following manner: Aliquots of fluid
from each vessel derived from the same pair of kidneys shall be pooled
and at least 10 milliliters of the pool inoculated into each system. The
dilution of the
[[Page 93]]
pool with medium shall be no greater than 1:4 and the area of surface
growth of cells shall be at least 3 square centimeters per milliliter of
test inoculum. The cultures shall be observed for at least 14 days. At
the end of the observation period, at least one subculture of fluid from
the Cercopithecus monkey kidney cell cultures shall be made in the same
tissue culture system and the subculture shall be observed for at least
14 days. If these tests indicate the presence in the monkey kidney
tissue culture production vessels of any viable microbial agent, the
viral harvest from these tissue cultures so implicated shall not be used
for poliovirus vaccine manufacture.
(4) Control vessels. At least 25 percent of the cell suspension from
each pair of kidneys shall be set aside and used to establish control
cultures. The control cultures shall be examined microscopically for
cell degeneration for an additional 14 days. The culture fluids from
such control cells shall be tested, both at the time of virus harvest
and at the end of the additional observation period, by the method
prescribed for testing of fluids in paragraph (b)(3) of this section. In
addition, the control cell sheet shall be examined for presence of
hemadsorbing viruses by the addition of guinea pig red blood cells.
(5) Interpretation of test results. At least 80 percent of the
control vessels shall be free of cell degeneration at the end of the
observation period to qualify the kidneys for poliovirus vaccine
manufacture. If the test results of the control cells indicate the
presence of any extraneous agent at the time of virus harvest, the virus
harvest from that tissue culture preparation shall not be used for
poliovirus vaccine manufacture. If any of the tests or observations
described in paragraph (b)(3) or (b)(4) of this section demonstrate the
presence in the tissue culture preparation of any microbial agent known
to be capable of producing human disease, the virus grown in each tissue
culture preparation shall not be used for poliovirus vaccine
manufacture.
(6) Temperature of kidney tissue production vessels after virus
inoculation. After virus inoculation, production vessels shall be
maintained at 33.0 to 35.0 deg.C during the course of virus
propagation.
(7) Kidney tissue virus harvests. Virus shall be harvested not later
than 72 hours after virus inoculation. Virus harvested from vessels
containing the kidney tissue from one monkey may be tested separately,
or samples of viral harvests from more than one pair of kidneys may be
combined, identified, and tested as a monovalent virus pool. Each pool
shall be mixed thoroughly and samples withdrawn for testing as
prescribed in Sec. 630.18(a). The samples shall be withdrawn immediately
after harvesting and prior to further processing, except that samples of
test materials frozen immediately after harvesting and maintained at -60
deg.C or below, may be tested upon thawing, provided no more than one
freeze-thaw cycle is employed.
(8) Filtration. After harvesting and removal of samples for the
safety tests prescribed in Sec. 630.18(a), the pool shall be passed
through sterile filters having a sufficiently small porosity to assure
bacteriologically sterile filtrates.
[56 FR 21432, May 8, 1991, as amended at 58 FR 19609, Apr. 15, 1993]
Sec. 630.14 Reference virus preparations.
(a) Titration test controls. The following reference viruses may be
obtained from the Center for Biologics Evaluation and Research:
(1) Reference Poliovirus, Live, Attenuated, Type 1, as a control for
correlation of virus titers in tissue cultures.
(2) Reference Poliovirus, Live, Attenuated, Type 2, as a control for
correlation of virus titers in tissue cultures.
(3) Reference Poliovirus, Live, Attenuated, Type 3, as a control for
correlation of virus titers in tissue cultures.
(4) Reference Poliovirus, Live, Attenuated, Trivalent, as a control
for correlation of virus titers in tissue cultures.
(b) Neurovirulence test controls. (1) Except as provided in
paragraph (b)(2) of this section, the following reference virus may be
obtained from the Center for Biologics Evaluation and Research:
[[Page 94]]
(i) Reference Attenuated Poliovirus, Type 1, as a control for
evaluation of monkey neurovirulence tests.
(ii) Reference Attenuated Poliovirus, Type 2, as a control for
evaluation of monkey neurovirulence tests.
(iii) Reference Attenuated Poliovirus, Type 3, as a control for
evaluation of monkey neurovirulence tests.
(2) Alternatively, upon FDA approval, World Health Organization
(WHO) reference standards of the corresponding type, WHO/I, WHO/II, and
WHO/III, may be used as controls for evaluation of monkey neurovirulence
tests.
Sec. 630.15 Potency test.
(a) Test for virus titer. The concentration of living virus in each
monovalent virus pool and in each trivalent vaccine, expressed as
infectivity titer per milliliter for cell cultures, shall be determined
using the Reference Poliovirus, Live, Attenuated of the same type as a
control or using another reference preparation of the same type that has
been calibrated against the appropriate reference preparation listed in
Sec. 630.14(a). A titration of the monovalent virus pool or the
trivalent vaccine shall not constitute a valid test unless the titration
of the reference virus when tested in parallel is within 0.5
log10 of its established titer. The titration of the parallel
reference is intended to validate the test system and shall not be used
to adjust the titer of the pool or lot under test.
(b) Dose. The human dose of trivalent vaccines shall be constituted
to have infectivity titers in the final container material of 106.0
to 107.0 for type 1, 105.1 to 106.1 for type 2, and
105.8 to 106.8 for type 3, when assayed in HEp-2 cells, or the
equivalent when titrated by a different method.
Sec. 630.16 Test for neurovirulence.
(a) Except as provided in Sec. 630.17, the following test relating
to safety prescribed in paragraph (b) of this section shall be performed
on each monovalent virus pool after the filtration process.
(b) Neurovirulence in monkeys. Except as provided in paragraph
(b)(5) of this section, each monovalent virus pool shall be tested
concurrently with the corresponding type Reference Attenuated Poliovirus
for neurovirulence by the intraspinal route of injection in Macaca
monkeys. Whenever possible the monkeys should be of comparable age and
weight and from the same quarantine group. The monkeys shall be
distributed randomly between the two test groups. If the number of
monkeys included in both groups precludes completion during a single
workday, approximately equal numbers of monkeys shall be inoculated with
the monovalent virus pool and the reference preparation during each of
the testing days. A preinjection serum sample obtained from each monkey
shall be shown to contain no neutralizing antibody in a dilution of 1:4
when tested against no more than 1,000 TCID50 (mean tissue culture
infectious doses) of each of the three types of poliovirus. The
neurovirulence test is not valid unless the inoculation sample is shown
to contain the equivalent of 106.5 to 107.5 TCID50 per
milliliter when a representative sample of the monovalent virus pool is
titrated in HEp-2 cells in comparison with the Reference Poliovirus,
Live, Attenuated of the appropriate type. All monkeys shall be observed
for 17 to 21 days and any evidence of physical abnormalities indicative
of poliomyelitis or other viral infections shall be recorded.
(1) Intraspinal inoculation. For tests with type 1 and type 2
monovalent virus pools and the Reference Attenuated Poliovirus of the
corresponding types, each of a group of at least 12 monkeys after being
suitably anesthetized shall be injected intraspinally into the
enlargement of the lumbar cord with 0.1 milliliter of the inoculation
sample. For tests with type 3 poliovirus materials, groups of at least
20 monkeys shall be injected as above after being suitably anesthetized.
A test of a virus pool shall include at least one group of monkeys, and
no more than three groups shall be inoculated, with the results from
testing one, two, or three groups of monkeys being evaluated as
prescribed in Sec. 630.16(b)(2). In addition, if on examination there is
no evidence of correct inoculation, additional animals may be inoculated
in order to reestablish the minimum number of 11 positive monkeys for
tests of types 1 and 2 virus
[[Page 95]]
pools and the minimum number of 18 positive monkeys for tests of Type 3
virus pools. A positive monkey is an animal which either survives for 11
or more days or succumbs or is sacrificed due to a severe poliovirus
infection at any time before the 11th day of the observation period and
in which neural lesions specific for poliovirus are seen in the central
nervous system. If at least 60 percent of the animals of a group survive
48 hours after inoculation, those animals that did not survive may be
replaced by additional animals. If less than 60 percent of the animals
in a group survive 48 hours after inoculation, the test shall be
considered invalid and shall be repeated.
(2) Determination of neurovirulence. At the conclusion of the
observation period, the animals are sacrificed and a comparative
evaluation shall be made of the evidence of neurovirulence of the
monovalent virus pool under test and the Reference Attenuated Poliovirus
of the corresponding type with respect to the histopathology of lesions
caused by poliovirus. Animals dying or sacrificed when severely
paralyzed or moribund during the test period, should be included in the
evaluation, except that these examinations of these monkeys shall be
made immediately after death. Histopathological examinations by a
qualified pathologist shall be made of at least the lumbar and cervical
enlargements, the medulla, the mesencephalon, the thalamus, and motor
cortex of each monkey in the groups injected with the monovalent virus
pool or with the reference under test. The magnitude of the
neuropathology exhibited in the lumbar and cervical areas, the medulla,
and mesencephalon of all positive monkeys inoculated with the monovalent
virus pool shall be quantified and compared to the magnitude of the
neuropathology determined based on the same type of evaluation of
monkeys in the current test and all previous tests of the Reference
Attenuated Poliovirus of the corresponding type. The monovalent virus
pool may be used for poliovirus vaccine if a comparative analysis of the
test results demonstrates that the numerical value assigned for
neurovirulence of the monovalent virus pool is equal to or less than
that of the Reference Attenuated Poliovirus of the corresponding type.
If the numerical value assigned for neurovirulence of the monovalent
virus pool is greater than that of the Reference Atteneuated Poliovirus,
the monovalent virus pool is acceptable if the difference is not greater
than that calculated by a mathematical method that is expected to reject
vaccines with neurovirulence identical to the reference at a frequency
of not less than 1 in 100 when 1 group of monkeys is inoculated. If 2
groups are injected with the same monovalent virus pool under test, the
frequency of rejection shall be not less than 5 in 100 and for 3 groups,
not less than 10 in 100. If the difference in numerical values is
greater than that calculated, irrespective of which reference
preparation was used in the test, the monovalent virus pool shall be
considered unacceptable and shall not be used for vaccine manufacture.
(3) Outlier scores. In the event that one or more monkeys inoculated
with virus from the monovalent virus pool have individual mean lesion
scores higher than that previously or concurrently associated with the
Reference Attenuated Poliovirus of the corresponding type, but the
monovalent virus pool meets the criteria for acceptable neurovirulence
given in Sec. 630.16(b)(2), the significance of the outlier scores shall
be evaluated by a method approved by the Director, Center for Biologics
Evaluation and Research before the vaccine may be released for use.
(4) Test with Reference Attenuated Poliovirus. Except as provided in
paragraph (b)(5) of this section, the Reference Attenuated Poliovirus of
the appropriate type shall be tested as prescribed in paragraph
(b)(1)(i) of this section concurrently with the monovalent virus pool.
More than one monovalent virus pool of the same type may be tested with
the same corresponding Reference Attenuated Poliovirus. Initially, a
minimum of four tests by the testing laboratory of each Reference
Attenuated Poliovirus is required. These tests must be such as to
provide sufficient experience to define the performance of the Reference
Attenuated Poliovirus and establish the variability of the assay. Each
test of
[[Page 96]]
the Reference Attenuated Poliovirus shall be considered acceptable and
added to the previous testing experience only if the magnitude of its
poliovirus neuropathology is statistically compatible with the results
of all previous tests with the same reference preparations of the same
type performed by the testing laboratory.
(5) Alternative procedures in case of monkey shortage. In the event
of a shortage of test monkeys and upon approval of the Director, Center
for Biologics Evaluation and Research, a monovalent virus pool may be
tested without concurrent testing of the corresponding type Reference
Attenuated Poliovirus. In such a case, the magnitude of the
neuropathology of the monovalent virus pool shall be compared with the
magnitude of the neuropathology exhibited in all previous tests of the
corresponding Reference Attenuated Poliovirus.
Sec. 630.17 Alternative test for neurovirulence.
(a) In lieu of the neurovirulence test in Sec. 630.16, the following
test may be performed after the filtration process, on each monovalent
virus pool or on each multiple thereof (monovalent lot).
(b) Neurovirulence in monkeys. Each monovalent virus pool or
monovalent lot shall be tested in comparison with the Reference
Attenuated Poliovirus, Type 1, for neurovirulence in Macaca monkeys by
both the intrathalamic and intraspinal routes of injection. A
preinjection serum sample obtained from each monkey must be shown to
contain no neutralizing antibody in a dilution of 1:4 when tested
against no more than 1,000 TCID50 (mean tissue culture infectious
dose) of each of the three types of poliovirus. The neurovirulence tests
are not valid unless the sample contains at least 107.6 TCID50
per milliliter when titrated in HEp-2 cells in comparison with the
Reference Poliovirus, Live, Attenuated of the appropriate type. All
monkeys shall be observed for 17 to 21 days and any evidence of physical
abnormalities indicative of poliomyelitis or other viral infections
shall be recorded.
(1) Intrathalamic inoculation. Each of at least 30 monkeys shall be
injected intracerebrally by placing 0.5 milliliter of virus pool
material into the thalamic region of each hemisphere. Comparative
evaluations shall be made with the virus pool under test and the
Reference Attenuated Poliovirus, Type 1. Only monkeys that show evidence
of inoculation into the thalamus shall be considered as having been
injected satisfactorily. With respect to inoculation, a test is deemed
valid if at least 24 monkeys are considered as having been injected
satisfactorily. If on examination there is evidence of failure to
inoculate virus pool material into the thalamus, additional monkeys may
be inoculated in order to reestablish the minimum number of monkeys for
the test.
(2) Intraspinal inoculation. Each of a group of at least five
monkeys shall be injected intraspinally with 0.2 milliliter of virus
pool material containing at least 107.6 TCID50 per milliliter
when titrated in HEp-2 cells, and each monkey in additional groups of at
least five monkeys shall be injected intraspinally with 0.2 milliliter
of a 1:1,000 and 1:10,000 dilution, respectively, of the same virus pool
material. Comparative evaluations shall be made with the virus pool
under test and the reference material. Only monkeys that show
microscopic evidence of inoculation into the gray matter of the lumbar
cord shall be considered as having been injected satisfactorily. With
respect to inoculation, a test is deemed valid if at least four monkeys
per group are considered as having been injected satisfactorily. If on
examination there is evidence of failure to inoculate intraspinally,
additional animals may be inoculated in order to reestablish the minimum
number of animals per group.
(3) Determination of neurovirulence. At the conclusion of the
observation period comparative histopathological examinations by a
qualified pathologist shall be made of the lumbar cord, cervical cord,
lower medulla, upper medulla, mesencephalon and motor cortex of each
monkey in the groups injected with virus under test and those injected
with the Reference Attenuated Poliovirus, Type 1, except that for
animals dying during the test period, these examinations shall be made
immediately after death. If at least 60
[[Page 97]]
percent of the animals of a group survive 48 hours after inoculation,
those animals which did not survive may be replaced by an equal number
of animals tested as prescribed in paragraph (b) of this section. If
less than 60 percent of the animals of a group survive 48 hours after
inoculation, the test must be repeated. At the conclusion of the
observation the animals shall be examined to ascertain whether the
distribution and histological nature of the lesions are characteristics
of poliovirus infection. A comparative evaluation shall be made of the
evidence of neurovirulence of the virus under test and the Reference
Attenuated Poliovirus, Type 1, with respect to:
(i) The number of animals showing lesions characteristic of
poliovirus infection;
(ii) The number of animals showing lesions other than those
characteristic of poliovirus infection;
(iii) The severity of the lesions;
(iv) The degree of dissemination of the lesions; and
(v) The rate of occurrence of paralysis not attributable to the
mechanical injury resulting from inoculation trauma. These five factors
may be weighted and interpreted as the Director, Center for Biologics
Evaluation and Research, or the Director's delegatees deem appropriate.
Among permissible interpretations, the factors may be considered in
different ways for monkeys inoculated intraspinally and for monkeys
inoculated intrathalamically. Other relevant factors in addition to
those listed in paragraph (b)(3)(i) through (b)(3)(v) of this section,
such as public health consequences, may be considered in evaluating
neurovirulence test results. The virus pool under test is satisfactory
for poliovirus vaccine only if at least 80 percent of the animals in
each group survive the observation period and if a comparative analysis
of the test results demonstrates that the neurovirulence of the test
virus pool does not exceed that of the Reference Attenuated Poliovirus,
Type 1.
(4) Test with Reference Attenuated Poliovirus. The Reference
Attenuated Poliovirus, Type 1, shall be tested as prescribed in
paragraphs (b)(1) and (b)(2) of this section at least once for every 10
production lots of vaccine, except that the interval between the test of
the reference and the test of any lot of vaccine shall not be greater
than 3 months. The test procedure shall be considered acceptable only if
lesions of poliomyelitis are seen in monkeys inoculated with the
reference material at a frequency statistically compatible with all
previous tests with this preparation.
Sec. 630.18 Additional tests for safety.
(a) Tests prior to filtration. Monovalent virus pools shall contain
no demonstrable viable microbial agent, except for unavoidable
bacteriophage and the intended attenuated live poliovirus. The vaccine
shall be tested for the absence of other infectious agents, including
polioviruses of other types or strains. Testing of each monovalent pool
shall include the following procedures:
(1) Inoculation of rabbits. A minimum of 100 milliliters of each
monovalent virus pool shall be tested by inoculation into at least 10
healthy rabbits, each weighing 1,500 to 2,500 grams. Each rabbit shall
be injected with a total of 1.0 milliliter intradermally in multiple
sites, and subcutaneously with 9.0 milliliters, of the monovalent virus
pool and the animals observed for at least 3 weeks. Each rabbit that
dies after the first 24 hours of the test, or is sacrificed because of
illness, shall be necropsied and the brain and organs removed and
examined. The monovalent virus pool may be used for poliovirus vaccine
only if at least 80 percent of the rabbits remain healthy and survive
the entire period and if all the rabbits used in the test fail to show
lesions of any kind at the sites of inoculation and fail to show
evidence of cercopithecid herpesvirus 1 or any other viral infection.
(2) Inoculation of adult mice. Each of at least 20 adult mice, each
weighing 15 to 20 grams, shall be inoculated intraperitoneally with 0.5
milliliter and intracerebrally with 0.03 milliliter of each monovalent
virus pool. The mice shall be observed for 21 days. Each mouse that dies
after the first 24 hours of the test, or is sacrificed because of
illness, shall be necropsied and
[[Page 98]]
examined for evidence of viral infection by direct observation and
subinoculation of appropriate tissue into at least five additional mice
which shall be observed for 21 days. The monovalent virus pool may be
used for poliovirus vaccine only if at least 80 percent of the mice
remain healthy and survive the entire period and if all the mice used in
the test fail to show evidence of lymphocytic choriomeningitis virus or
other viral infection.
(3) Inoculation of suckling mice. Each of at least 20 suckling mice
less than 24 hours old shall be inoculated intracerebrally with 0.01
milliliter and intraperitonally with 0.1 milliliter of the monovalent
virus pool. The mice shall be observed daily for at least 14 days. Each
mouse that dies after the first 24 hours of the test, or is sacrificed
because of illness, shall be necropsied and examined for evidence of
viral infection. Such examination shall include subinoculation of
appropriate tissue suspensions into an additional group of at least five
suckling mice by the intracerebral and intraperitoneal routes and
observed daily for 14 days. In addition, a blind passage shall be made
of a single pool of the emulsified tissue (minus skin and viscera) of
all mice surviving the original 14-day test. The monovalent virus pool
may be used for poliovirus vaccine only if at least 80 percent of the
mice remain healthy and survive the entire period and if all the mice
used in the test fail to show evidence of Coxsackie or other viral
infection.
(4) Inoculation of guinea pigs. Each of at least five guinea pigs,
each weighting 350 to 450 grams, shall be inoculated intracerebrally
with 0.1 milliliter and intraperitoneally with 5.0 milliliters on the
monovalent virus pool to be tested. The animals shall be observed for at
least 42 days and rectal temperatures recorded daily for the last 3
weeks of the test. Each animal that dies after the first 24 hours of the
test, or is sacrificed because of illness, shall be necropsied and its
tissues shall be examined both microscopically and culturally for
evidence of tubercle bacilli, and by passage of tissue suspensions into
at least three other guinea pigs by the intracerebral and
intraperitoneal routes of inoculation for evidence of viral infection.
If clinical signs suggest infection with lymphocytic choriomeningitis
virus, serological tests shall be performed on blood samples of the test
guinea pigs to confirm the clinical observations. Animals that die or
are sacrificed during the first 3 weeks after inoculation with the
monovalent virus pools shall be examined for infection with lymphocytic
choriomeningitis virus. Animals that die in the final 3 weeks shall be
examined both microscopically and culturally for Mycobacterium
tuberculosis. The monovalent virus pool may be used for poliovirus
vaccine only if at least 80 percent of all animals remain healthy and
survive the observation period and if all the animals used in the test
fail to show evidence of infection with Mycobacterium tuberculosis or
any viral infection.
(5) Inoculation of monkey kidney tissue cultures. At least 500 doses
or 50 milliliters, whichever is a greater volume of virus, taken either
from each undiluted monovalent virus pool or, in equal proportions from
individual harvests or subpools, shall be tested for simian viruses in
Macaca monkey kidney tissue cultures and, in the same volume, in
Cercopithecus monkey kidney tissue cultures. A dilution of the virus
pool in medium not to exceed 1:4 shall be used. The area of surface
growth of the cells shall be at least 3 square centimeters per
milliliter of test inoculum. The test poliovirus shall be neutralized by
high-titer specific antiserum of nonprimate origin. The immunizing
antigens used for the preparation of antisera shall be grown in a cell
line other than the cell line used for testing the vaccine. The cultures
shall be observed for at least 14 days. At the end of the observation
period at least one subculture of fluid from the Cercopithecus kidney
cell culture shall be made in the same tissue culture system and the
subculture shall be observed for at least 14 days. The monovalent virus
pool may be used for poliovirus vaccine only if all the tissue cultures
fail to show evidence of the presence of simian viruses or any other
viral infection.
(6) Inoculation of human cell cultures. At least 500 doses or 50
milliliters, whichever represents a greater volume of virus, taken from
either a single monovalent
[[Page 99]]
pool or, in equal proportions from individual harvests or subpools,
shall be tested for the presence of measles virus in either:
(i) Primary human amnion cells,
(ii) Primary human kidney cells, or
(iii) Any other human or nonhuman cell system of comparable
suspectibility to unmodified measles virus.
The virus pool shall be diluted with medium not to exceed 1:4. The area
of surface growth of cells shall be at least 3 square centimeters per
milliliter of test inoculum. The test material shall be neutralized with
poliovirus antiserum of other than primate origin if the tissue culture
cell system used is susceptible to poliovirus. The immunizing antigens
used for the preparation of antiserum shall be grown in a cell line
other than the cell line used for testing the vaccine. The culture shall
be observed for at least 14 days. The monovalent virus pool may be used
for poliovirus vaccine only if all tissue cultures fail to show evidence
of the presence of measles virus or any other viral infection.
(7) Inoculation of a rabbit kidney tissue culture. At least 500
milliliters of virus pool, taken from either a single monovalent pool or
in equal proportions from individual harvests or subpools, shall be
tested in primary rabbit kidney tissue culure preparations for evidence
of cercopithecid herpesvirus 1. The virus pool shall be diluted with
medium not to exceed 1:4. The area of surface growth of cells shall be
at least 3 square centimeters per milliliter of test inoculum. The
culture shall be observed for at least 14 days. The monovalent virus
pool may be used for poliovirus vaccine only if all tissue cultures fail
to show evidence of the presence of herpesvirus.
(b) Tests for in vitro markers. In addition to the neurovirulence
test required by Secs. 630.16 or 630.17, the following tests relating to
safety shall be performed on each monovalent virus pools after the
filtration process. Tests shall be performed on each monovalent virus
pool using the marker tests described below or other methods shown to be
of comparable value in indentification of the attenuated strain. The
test results shall demonstrate that the monovalent virus pool under test
and the seed virus have substantially the same marker characteristics.
(1) rct/40 Marker. Attenuated strains which grow readily at 40
deg.C (0.5 deg.C) are classified as rct/40 positive (+) in
contrast to the rct/40 negative (-) strains, which show an increased
growth of at least 100,000 fold at 36 deg.C over that obtained at 40
deg.C. Comparative determinations shall be made in suitable culture
vessels.
(2) d Marker. Attenuated strains which grow readily at low
concentrations of bicarbonate under agar are classified as d positive
(+) in contrast to the d negative (-) strains, which exhibit delayed
growth under the same conditions. The cultures shall be grown in a 36
deg.C incubator, in suitable culture vessels in an environment of 5
percent CO2 in air.
(c) Final container sterility test. The final container sterility
test need not be performed provided aseptic techniques are used in the
filling process.
[56 FR 21432, May 8, 1991; 56 FR 27787, June 17, 1991]
Sec. 630.19 General requirements.
(a) Vaccine release. No lot of trivalent vaccine shall be released
by the manufacturer unless each monovalent virus pool contained therein:
(1) Has been manufactured by the same procedures;
(2) Has met the criteria of neurovirulence for monkeys prescribed in
Secs. 630.16(b) or 630.17(b);
(3) Has met the criteria of in vitro markers prescribed in
Sec. 630.18(b); and
(4) Has been released for further manufacturing by the Director,
Center for Biologics Evaluation and Research unless, at the Director's
discretion, the Director determines that lot release by the Center for
Biologics Evaluation and Research is not required. The protocols for all
monovalent virus pools produced sequentially from the same seed and
tested, in whole or in part, in accordance with Secs. 630.16(b) or
630.17(b) shall be submitted to the Director, Center for Biologics
Evaluation and Research, whether or not release of the pool for further
manufacturing is requested. For monovalent virus pools not tested under
Secs. 630.16(b) or 630.17(b),
[[Page 100]]
the manufacturer shall report the reasons for partial manufacture to the
Director, Center for Biologics Evaluation and Research.
(b) Labeling. In addition to the items required by other applicable
labeling provisions of this chapter, the final container label shall
bear a statement indicating that liquid vaccine may not be used for more
than 7 days after opening the container. Labeling may include a
statement indicating that, for frozen vaccine, a maximum of 10 freeze-
thaw cycles is permissible provided the total cumulative duration of
thaw does not exceed 24 hours, and provided the temperature does not
exceed 8 deg.C during the periods of thaw.
(c) Samples and protocols. For each trivalent lot of vaccine and for
each monovalent virus pool, the following materials shall be submitted
in accordance with instructions received from the Director, Center for
Biologics Evaluation and Research, 8800 Rockville Pike, Bethesda, MD
20892.
(1) A protocol that consists of a summary of the history of
manufacture of each trivalent lot or monovalent virus pool, including
any test results requested by the Director, Center for Biologics
Evaluation and Research.
(2) Twenty milliliters of monovalent virus pool before filtration.
(3) Forty milliliters of monovalent virus pool after filtration. The
titer of the sample shall be no less than the equivalent of 10 7.5
TCID 50 per milliliter when titrated in HEp-2 cells; if the titer
is greater than 10 7.5 TCID 50 per milliliter, a
correspondingly smaller volume may be submitted.
(4) A total of at least 50 single doses or the equivalent thereof of
the trivalent vaccine.
(5) When deemed appropriate, the Director, Center for Biologics
Evaluation and Research, may require submission of samples or sample
volumes other than those specified in paragraphs (c)(2), (c)(3), and
(c)(4) of this section.
(d) Public health implications. In interpreting any provision of the
regulations governing oral poliovirus vaccine, the agency may consider
any potential effect on individual or public health, including effects
related to vaccine supply.
(e) Alternative procedures. (1) The Director, Center for Biologics
Evaluation and Research, may approve an exception or alternative to any
requirement in subpart B of part 630 regarding Poliovirus Vaccine Live
Oral. Requests for such exceptions or alternatives should ordinarily be
made in writing. However, in limited circumstances such requests may be
made orally and permission may be given orally by the Director, Center
for Biologics Evaluation and Research. Oral requests and approvals must
be followed by written requests and written approvals.
(2) FDA will publish a list of approved alternative procedures and
exceptions periodically in the Federal Register.
(f) Status of vaccine in distribution. Poliovirus Vaccine Live Oral
released or in distribution prior to May 8, 1991, is deemed to meet the
requirements of supart B of part 630.
Subpart C--[Reserved]
Subpart D--Measles Virus Vaccine Live
Sec. 630.30 Measles Virus Vaccine Live.
(a) Proper name and definition. The proper name of this product
shall beMeasles Virus Vaccine Live, which shall consist of a preparation
of live, attenuated, measles virus.
(b) Criteria for acceptable strains of attenuated measles virus.
Strains of attenuated measles virus used in the manufacture of vaccine
shall be identified by (1) historical records, including origin and
manipulation during attenuation and (2) antigenic specificity as measles
virus as demonstrated by tissue culture neutralization tests. Strains
used for the manufacture ofMeasles Virus Vaccine Live, shall have been
shown to be safe and potent in man by field studies with experimental
vaccines. The vaccine shall have been demonstrated as safe and potent in
at least 10,000 susceptible persons. Susceptibility shall be shown by
the absence of neutralizing or other antibodies against measles virus,
or by other appropriate methods. Seed virus used for vaccine manufacture
shall be free of all demonstrable extraneous
[[Page 101]]
viable microbial agents except for unavoidable bacteriophage.
(c) Neurovirulence safety test of the virus seed strain in monkeys--
(1) The test. A demonstration shall be made in monkeys of the lack of
neurotropic properties of the seed strain of attenuated measles virus
used in the manufacture of measles virus vaccine. For this purpose and
to establish consistency of manufacture of the vaccine, vaccine from
each of five consecutive lots shall be tested separately in the
following manner:
(i) Samples of each of the five lots of vaccine shall be tested in
measles susceptible monkeys. Immediately prior to initiation of a test
each monkey shall have been shown to be serologically negative for
neutralizing antibodies by means of a tissue culture neutralization test
with undiluted serum from each monkey tested at approximately 100
TCID50 of Edmonston strain measles virus, or negative for measles
virus antibodies as demonstrated by tests of equal sensitivity.
(ii) A test sample of vaccine removed after clarification but before
final dilution for standardization of virus content shall be used for
the test.
(iii) Vaccine shall be injected by combined intracerebral,
intraspinal, and intramuscular routes into not less than 20 Macaca or
Cercopithecus monkeys or a species found by the Director, Center for
Biologics Evaluation and Research, to be equally suitable for the
purpose. The animals shall be in overt good health and injected under
deep barbiturate anesthesia. The intramuscular injection shall consist
of 1.0 milliliter of test sample into the right leg muscles. At the same
time, 200 milligrams of cortisone acetate shall be injected into the
left leg muscles, and 1.0 milliliter of procaine penicillin (300,000
units) into the right arm muscles. The intracerebral injection shall
consist of 0.5 milliliter of test sample into each thalamic region of
each hemisphere. The intraspinal injection shall consist of 0.5
milliliter of test sample into the lumber spinal cord enlargement.
(iv) The monkeys shall be observed for 17-21 days and symptoms of
paralysis as well as other neurologic disorders shall be recorded.
(v) At least 90 percent of the test animals must survive the test
period without losing more than 25 percent of their weight except that,
if at least 70 percent of the test animals survive the first 48 hours
after injection, those animals which do not survive this 48-hour test
period may be replaced by an equal number of qualified test animals
which are tested pursuant to paragraphs (c)(1)(i) through (iv) of this
section. At least 80 percent of the injected animals surviving beyond
the first 48 hours must show gross or microscopic evidence of
inoculation trauma in the thalamic area and microscopic evidence of
inoculation trauma in the lumbar region of the spinal cord. If less than
70 percent of the test animals survive the first 48 hours, or if less
than 80 percent of the animals meet the inoculation criteria prescribed
in this paragraph, the test must be repeated.
(vi) At the end of the observation period, each surviving monkey
shall (a) be bled and the serum tested for evidence of serum antibody
conversion to measles virus and (b) be autopsied and samples of cerebral
cortex and of cervical and lumbar spinal cord enlargements shall be
taken for virus recovery and identification if needed pursuant to
paragraph (c)(1)(vii) of this section. Histological sections shall be
prepared from both spinal cord enlargements and appropriate sections of
the brain and examined.
(vii) Doubtful histopathological findings necessitate (a)
examination of a sample of sections from several regions of the brain in
question, and (b) attempts at virus recovery from the nervous systems
tissues previously removed from the animal.
(viii) The lot is satisfactory if the histological and other studies
demonstrate no evidence of changes in the central nervous system
attributable to unusual neurotropism of the seed virus or of the
presence of extraneous neurotropic agents.
(2) Wild virus controls. As a check against the inadvertent
introduction of wild measles virus, at least four uninoculated measles
susceptible control monkeys shall be maintained as either cage mates to,
or within the same immediate area of, the 20 inoculated test animals for
each lot of vaccine for
[[Page 102]]
the entire period of observation (17-21 days) and an additional 10 days.
Serum samples from these control contact monkeys drawn at the time of
seed virus inoculation of the test animals, and again after completion
of the test, shall be shown to be free of measles neutralizing
antibodies.
(3) Test results. (i) For each lot of vaccine under test, at least
80 percent of the monkeys must show measles antibody serological
conversion (1:4 or greater) when the serum as obtained from the monkey
is tested and the control contact monkeys must demonstrate no
immunological response indicative of measles virus infection.
(ii) The measles virus seed has acceptable neurovirulence properties
for use in vaccine manufacture only if for each of the five lots (a) 90
percent of the monkeys survive the observation period, (b) the
histological and other studies produce no evidence of changes in the
central nervous system attributable to unusual neurotropism of the seed
virus, and (c) there is no evidence of the presence of extraneous
neurotropic agents.
(4) Need for additional neurovirulence safety testing. A
neurovirulence safety test as prescribed in this paragraph shall be
performed on vaccine from five consecutive lots whenever a new
production seed lot is introduced or whenever the source of cell culture
substrate must be reestablished and recertified as prescribed in
Sec. 630.32(a) and (b) of this part.
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
49 FR 23834, June 8, 1984; 50 FR 4138, Jan. 29, 1985; 55 FR 11013, Mar.
26, 1990; 55 FR 47875, Nov. 16, 1990]
Sec. 630.31 Clinical trials to qualify for license.
To qualify for license, the antigenicity of the vaccine shall have
been determined by clinical trials of adequate statistical design, by a
suitable route of administration of the product. Such clinical trials
shall be conducted with five lots of measles virus vaccine which have
been manufactured by the same methods. There shall be a demonstration
under circumstances in which adequate clinical and epidemiological
surveillance of illness has been maintained to show that the measles
virus vaccine, when administered as recommended by the manufacturer, is
free of harmful effect upon administration to approximately 1,000
susceptible individuals, in that there were no detectable neutralizing
antibodies before vaccination and there was serological conversion after
vaccination. The five lots of vaccine shall be distributed as evenly as
possible among the 1,000 individuals tested. Demonstration shall be made
of immunogenic effect by the production of specific measles neutralizing
antibodies (i.e., sero-conversion from less than 1:4 to 1:8 or greater)
in at least 90 percent of each of five groups of measles susceptible
individuals, each having received a virus vaccine dose which is not
greater than that which was demonstrated to be safe in field studies
(Sec. 630.30(b)) when used under comparable conditions. Such clinical
trials shall be conducted in compliance with part 56 of this chapter
unless exempted under Sec. 56.104 or granted a waiver under Sec. 56.105,
and with the requirements for informed consent set forth in part 50 of
this chapter.
[55 FR 47875, Nov. 16, 1990]
Sec. 630.32 Manufacture of live, attenuated Measles Virus Vaccine.
(a) Virus cultures. Virus shall be propagated in chick embryo tissue
cultures.
(b) Virus propagated in chick embryo tissue cultures. Embryonated
chicken eggs used as the source of chick embryo tissue for the
propagation of measles virus shall be derived from flocks certified to
be free of Salmonella pullorum, avian tuberculosis, fowl pox, Rous
sarcoma, avian leucosis, reticuloendotheliosis virus, and other
adventitious agents pathogenic for chickens. If eggs are procured from
flocks that are not so certified, tests shall be performed to
demonstrate freedom of the vaccine from such agents. (See
Sec. 630.35(a)(8) for test for avian leucosis.)
(c) [Reserved]
(d) Passage of virus strain in vaccine manufacture. Virus in the
final vaccine shall represent no more than ten tissue culture passages
beyond the passage used to perform the clinical trials (Sec. 630.30(b))
which qualified the manufacturer's vaccine strain for license.
[[Page 103]]
(e) Tissue culture preparation. Only primary cell tissue cultures
shall be used in the manufacture of Measles Virus Vaccine. Continuous
cell lines shall not be introduced or propagated in Measles Virus
Vaccine manufacturing areas.
(f) Control vessels. (1) From the tissue used for the preparation of
tissue cultures for growing attenuated measles virus, an amount of
processed cell suspension equivalent to that used to prepare 500 ml. of
tissue culture shall be used to prepare uninfected tissue control
materials. This material shall be distributed in control vessels and
observed microscopically for a period of no less than 14 days beyond the
time of inoculation of the production vessels with measles virus; but if
the production vessels are held for use in vaccine manufacture for more
than 14 days, the control vessels shall be held and observed for the
additional period. At the end of the observation period or at the time
of virus harvest, whichever is later, fluids from the control cultures
shall be tested for the presence of adventitious agents as follows:
Samples of fluid from each control vessel shall be collected at the
same time as fluid is harvested from the corresponding production
vessels. If multiple virus harvests are made from the same cell
suspension, the control samples for each harvest shall be frozen and
stored at -60 deg. C. until the last viral harvest for that cell
suspension is completed. The fluid from all the control samples from
that suspension shall be pooled in proportionate amounts and at least
five ml. inoculated into human and simian cell tissue culture systems
and in the tissue culture system used for virus production. The cultures
shall be observed for the presence of changes attributable to growth of
adventitious viral agents including hemadsorption viral agents.
(2) The cell sheets of one quarter to one third of the control
vessels shall be examined at the end of the observation period (14 days
or longer) for the presence of hemadsorption viruses by the addition of
guinea pig red blood cells. If the chick embryo cultures were not
derived from a certified source (paragraph (b) of this section), the
remaining tissue culture controls may be used to test for avian leucosis
virus using either Rubin's procedure for detecting Resistance Inducing
Factor (RIF) or a method of equivalent effectiveness.
(3) The test is satisfactory only if there is no evidence of
adventitious viral agents and if at least 80 percent of the control
vessels are available for observation at the end of the observation
period (14 days or longer).
(g) Test samples. Samples of virus harvests or pools for testing by
inoculation into animals, into tissue culture systems, into embryonated
hens' eggs, and into bacteriological media, shall be withdrawn
immediately after harvesting or pooling but prior to freezing except
that samples of test materials frozen immediately after harvesting or
pooling and maintained at -60 deg. C. or below, may be tested upon
thawing, provided no more than two freeze-thaw cycles are employed. The
required tests shall be initiated without delay after thawing.
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
47 FR 24699, June 8, 1982]
Sec. 630.33 Reference virus.
A U.S. Reference Measles Virus, Live, Attenuated, shall be obtained
from the Center for Biologics Evaluation and Research as a control for
correlation of virus titers.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
Sec. 630.34 Potency test.
The concentration of live measles virus shall constitute the measure
of potency. The titration shall be performed in a suitable cell culture
system, free of wild viruses, using either the U.S. Reference Measles
Virus, Live, Attenuated or a calibrated equivalent strain as a titration
control. The concentration of live measles virus contained in the
vaccine of each lot under test shall be no less than the equivalent of
1,000 TCID50 of the U.S. reference per human dose.
Sec. 630.35 Test for safety.
(a) Tests prior to clarification of vaccine manufactured in chick
embryo tissue cultures. Prior to clarification, the following tests
shall be performed on each virus pool of chick embryo tissue culture:
[[Page 104]]
(1) Inoculation of adult mice. Each of at least 20 adult mice each
weighing 15-20 grams shall be inoculated intraperitoneally with 0.5 ml.
and intracerebrally with 0.03 ml. amounts of each virus pool to be
tested. The mice shall be observed for 21 days. Each mouse that dies
after the first 24 hours of the test, or is sacrificed because of
illness, shall be necropsied and examined for evidence of viral
infection by direct observation and subinoculation of appropriate tissue
into at least five additional mice which shall be observed for 21 days.
The virus pool may be used only if at least 80 percent of the original
group of mice remain healthy and survive the observation period and if
none of the mice show evidence of a transmissible agent or other viral
infection, other than measles virus, attributable to the vaccine.
(2) Inoculation of suckling mice. Each of at least 20 suckling mice
less than 24 hours old shall be inoculated intracerebrally with 0.01 ml.
and intraperitoneally with 0.1 ml. of the virus pool to be tested. The
mice shall be observed daily for at least 14 days. Each mouse that dies
after the first 24 hours of the test, or is sacrificed because of
illness, shall be necropsied and examined for evidence of viral
infection. Such examination shall include subinoculation of appropriate
tissue suspensions into an additional group of at least five suckling
mice by intracerebral and intraperitoneal routes and observed daily for
14 days. In addition, a blind passage shall be made of a single pool of
the emulsified tissue (minus skin and viscera) of all mice surviving the
original 14-day test. The virus pool is satisfactory for Measles Virus
Vaccine only if at least 80 percent of the original inoculated mice
remain healthy and survive the entire observation period, and if none of
the mice used in the test show evidence of a transmissible agent or
viral infection, other than measles virus, attributable to the vaccine.
(3) Inoculation of monkey tissue cell cultures. A volume of virus
suspension of each undiluted virus pool, equivalent to at least 500
human doses or 50 milliliters, whichever represents a greater volume,
shall be tested for adventitious agents in Cercopithecus monkey kidney
tissue culture preparations or Erythrocebus patas monkey kidney tissue
culture preparations, after neutralization of the measles virus by a
high titer antiserum of nonhuman, nonsimian and nonchicken origin. The
immunizing antigen used for the preparation of the measles antiserum
shall be grown in tissue culture cells that shall be free of extraneous
viruses which might elicit antibodies that could inhibit growth of
extraneous viruses present in the measles virus pool. The tissue culture
of the virus pool shall be observed for no less than 14 days. The virus
pool is satisfactory for measles virus vaccine only if all the tissue
culture tests fail to show evidence of any extraneous transmissible
agent other than measles virus attributable to the vaccine.
(4) Inoculation of other cell cultures. The measles virus pool shall
be tested in the same manner as prescribed in paragraph (a)(3) of this
section in rhesus or cynomolgus monkey kidney, chick embryo, and human
tissue cell cultures.
(5) Inoculation of embryonated chicken eggs. A volume of virus
suspension of each undiluted virus pool, equivalent to at least 100
doses or 10 milliliters, whichever represents a greater volume, after
neutralization of the measles virus by a high titer antiserum of
nonhuman, nonsimian, nonavian origin shall be tested as follows:
(i) Embryonated eggs, 10 to 11 days old, shall be inoculated by the
allantoic route using 0.5 milliliter per egg. Follow incubation at
35 deg. C for 72 hours, the allantoic fluids shall be harvested, pooled,
and subpassed by the same route into fresh, embryonated eggs, 10 to 11
days old, using 0.5 milliliter per egg and incubated at 35 deg. C for 72
hours. Both the initial pool and the subpassage harvest shall be tested
for the presence of hemagglutinin. The virus pool is satisfactory if the
embryos appear normal and there is no evidence of hemagglutinating
agents.
(ii) Embryonated eggs, 6 to 7 days old, shall be inoculated by the
yolk sac route using 0.5 milliliter per egg. Following incubation at
35 deg. C for at least 9 days, the yolk sacs shall be harvested and
pooled. A 10-percent suspension of
[[Page 105]]
yolk sacs shall be subpassed by the same route into fresh embryonated
eggs, 6 to 7 days old, using 0.5 milliliter of inoculum per egg and
incubated at 35 deg. C for at least 9 days. The virus pool is
satisfactory if the embryos in both the initial test and the subpassage
appear normal.
(6) [Reserved]
(7) Bacteriological tests. Each virus pool shall be tested for
sterility in accordance with Sec. 610.12 of this chapter. In addition
each virus pool shall be tested for the presence of M. tuberculosis,
both avian and human, by appropriate culture methods.
(8) Test for avian leucosis. If the cultures were not derived from a
certified source (Sec. 630.32(b)), and the control fluids were not
tested for avian leucosis (Sec. 630.32(f)), at least 500 doses or 50
ml., whichever represents a greater volume of each undiluted vaccine
pool, shall be tested and found negative for avian leucosis, using
either Rubin's procedure for detecting Resistance Inducing Factor (RIF)
or another method of equivalent effectiveness.
(b) [Reserved]
(c) Clarification. After harvesting and removal of samples for
testing as prescribed above in this section, the virus fluids shall be
clarified by centrifugation, by passage through filters of sufficiently
small porosity, or by any other method that will assure removal of all
intact tissue cells which may have been collected in the harvesting
process.
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
41 FR 43400, Oct. 1, 1976; 47 FR 24699, June 8, 1982]
Sec. 630.36 General requirements.
(a) Final container tests. In addition to the tests required
pursuant to Sec. 610.14 of this chapter, an immunological and
virological identity test shall be performed on the final container if
it was not performed on each pool or the bulk vaccine prior to filling.
(b)--(c) [Reserved]
(d) Dose. These standards are based on an individual human
immunizing dose of no less than 1,000 TCID50 of Measles Virus
Vaccine Live, expressed in terms of the assigned titer of the U.S.
reference measles virus.
(e) Labeling. In addition to the items required by other applicable
labeling provisions of this subchapter, single-dose container labeling
for vaccine which is not protected against photochemical deterioration
shall include a statement cautioning against exposure to sunlight.
(f) [Reserved]
(g) Photochemical deterioration; protection. Vaccine in multiple
dose final containers shall be protected against photochemical
deterioration. Such containers may be colored, or outside coloring or
protective covering may be used for this purpose, provided (1) the
method used is shown to provide the required protection, and (2) visible
examination of the contents is not precluded. Vaccine in single dose
containers may be protected in the same manner provided the same
conditions are met.
(h) Sample and protocols. The following materials shall be submitted
to the Director, Center for Biologics Evaluation and Research, Food and
Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892:
(1) For each lot of vaccine:
(i) A protocol which consists of a summary of the history of the
manufacture of the lot, including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research.
(ii) A total of no less than two 25-milliliter volumes in a frozen
state (-60 deg. C) of preclarification bulk vaccine containing no
preservative or adjuvant.
(iii) A total of no less than 30 containers of the vaccine from each
filling of each bulk lot of single-dose containers. A total of no less
than six 50-dose containers or ten 10-dose containers of the vaccine
from each filling of each bulk lot of multiple-dose containers.
(2) In addition to the requirements of paragraph (h)(1) of this
section, whenever a new production seed lot is introduced, or whenever
the source of cell culture substrate must be reestablished and
recertified, samples consisting of no less than 100 milliliters in 10
milliliter volumes, in a frozen state (-60 deg. C), of postclarification
bulk vaccine
[[Page 106]]
containing stabilizer but no preservative or adjuvant, taken from each
of 5 consecutive lots of the bulk vaccine.
[38 FR 32068, Nov. 20, 1973, as amended at 41 FR 10429, Mar. 11, 1976;
49 FR 23834, June 8, 1984; 50 FR 4138, Jan. 29, 1985; 51 FR 15610, Apr.
25, 1986; 55 FR 11013, Mar. 26, 1990]
Subpart E--[Reserved]
Subpart F--Mumps Virus Vaccine Live
Sec. 630.50 Mumps Virus Vaccine Live.
(a) Proper name and definition. The proper name of this product
shall be Mumps Virus Vaccine Live, which shall consist of a preparation
of live, attenuated mumps virus.
(b) Criteria for acceptable strains of attenuated mumps virus.
Strains of attenuated mumps virus used in the manufacture of vaccine
shall be identified by (1) historical records including origin and
manipulation during attenuation, (2) antigenic specificity as mumps
virus as demonstrated by tissue culture neutralization tests. Strains
used for the manufacture of Mumps Virus Vaccine Live shall have been
shown to be safe and potent in at least 5,000 susceptible individuals by
field studies with experimental vaccines. Susceptibility shall be shown
by the absence of neutralizing or other antibodies against mumps virus,
or by other appropriate methods. Seed virus used for vaccine manufacture
shall be free of all demonstrable extraneous viable microbial agents
except for unavoidable bacteriophage.
(c) Neurovirulence safety test of the virus seed strain in monkeys--
(1) The test. A demonstration shall be made in monkeys of the lack of
neurotropic properties of the seed strain of attenuated mumps virus used
in the manufacture of mumps vaccine. For this purpose and to establish
consistency of manufacture of the vaccine, vaccine from each of five
consecutive lots shall be tested separately in monkeys shown to be
serologically negative for mumps virus antibodies in the following
manner:
(i) A test sample of vaccine removed after clarification but before
final dilution for standardization of virus content shall be used for
the test.
(ii) Vaccine shall be injected by combined intracerebral,
intraspinal, and intramuscular routes into not less than 20 Macaca or
Cercopithecus monkeys or a species found by the Director, Center for
Biologics Evaluation and Research, to be equally suitable for the
purpose. The animals shall be in overt good health and injected under
deep barbiturate anesthesia. The intramuscular injection shall consist
of 1.0 milliliter of test sample into the right leg muscles. At the same
time, 200 milligrams of cortisone acetate shall be injected into the
left leg muscles, and 1.0 milliliter of procaine penicillin (300,000
units) into the right arm muscles. The intracerebral injection shall
consist of 0.5 milliliter of test sample into each thalamic region of
each hemisphere. The intraspinal injection shall consist of 0.5
milliliter of test sample into the lumbar spinal cord enlargement.
(iii) The monkeys shall be observed for 17-21 days and symptoms of
paralysis as well as other neurologic disorders shall be recorded.
(iv) At least 90 percent of the test animals must survive the test
period without losing more than 25 percent of their weight except that,
if at least 70 percent of the test animals survive the first 48 hours
after injection, those animals which do not survive this 48-hour test
period may be replaced by an equal number of qualified test animals
which are tested pursuant to paragraphs (c)(1)(i) through (iii) of this
section. At least 80 percent of the injected animals surviving beyond
the first 48 hours must show gross or microscopic evidence of
inoculation trauma in the thalamic area and microscopic evidence of
inoculation trauma in the lumbar region of the spinal cord. If less than
70 percent of the test animals survive the first 48 hours, or if less
than 80 percent of the animals meet the inoculation criteria prescribed
in this paragraph, the test must be repeated.
(v) At the end of the observation period, each surviving animal
shall be autopsied and samples of cerebral cortex and of cervical and
lumbar spinal cord enlargements shall be taken for virus recovery and
identification if
[[Page 107]]
needed pursuant to paragraph (c)(1) (vi) of this section. Histological
sections shall be prepared from both spinal cord enlargements and
appropriate sections of the brain and examined.
(vi) Doubtful histopathological findings necessitate (a) examination
of a sample of sections from several regions of the brain in question,
and (b) attempts at virus recovery from the nervous system tissues
previously removed from the animals.
(vii) The lot is satisfactory if the histological and other studies
demonstrate no evidence of changes in the central nervous system
attributable to unusual neurotropism of the seed virus or of the
presence of extraneous neurotropic agents.
(2) Test results. The mumps virus seed has acceptable neurovirulence
properties for use in vaccine manufacture only if for each of the five
lots (i) 90 percent of the monkeys survive the observation period, (ii)
the histological and other studies produce no evidence of changes in the
central nervous system attributable to unusual neurotropism or
replication of the seed virus and (iii) there is no evidence of the
presence of extraneous neurotropic agents.
(3) Need for additional neurovirulence safety testing. A
neurovirulence safety test as prescribed in this paragraph shall be
performed on vaccine from five consecutive lots whenever a new
production seed lot is introduced or whenever the source of cell culture
substrate must be reestablished and recertified as prescribed in
Sec. 630.52(a).
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 50
FR 4138, Jan. 29, 1985; 55 FR 11013, Mar. 26, 1990; 55 FR 47875, Nov.
16, 1990]
Sec. 630.51 Clinical trials to qualify for license.
To qualify for license, the antigenicity of Mumps Virus Vaccine Live
shall be determined by clinical trials, conducted in compliance with
part 56 of this chapter unless exempted under Sec. 56.104 or granted a
waiver under Sec. 56.105, and with part 50 of this chapter, that follow
the procedures prescribed in Sec. 630.31, except that the immunogenic
effect shall be demonstrated by establishing that a protective antibody
response has occurred in at least 90 percent of each of the five groups
of mumps-susceptible individuals, each having received the parenteral
administration of a virus vaccine dose not greater than that
demonstrated to be safe in field studies (Sec. 630.50(b)) when used
under comparable conditions.
[46 FR 8956, Jan. 27, 1981, as amended at 50 FR 4138, Jan. 29, 1985]
Sec. 630.52 Manufacture of Mumps Virus Vaccine Live
(a) Virus cultures. Mumps virus shall be propagated in chick embryo
cell cultures. The embryonated chicken eggs used as the source of chick
embryo tissue for the propagation of mumps virus shall be derived from
flocks certified or tested as prescribed in Sec. 630.32(b).
(b) Passage of virus strain in vaccine manufacture. Virus in the
final vaccine shall represent no more than five cell culture passages
beyond the passage used to perform the clinical trials (Sec. 630.50(b))
which qualified the manufacturer's vaccine strain for license.
(c) Cell culture preparation. Only primary cell cultures shall be
used in the manufacture of mumps virus vaccine. Continuous cell lines
shall not be introduced or propagated in mumps virus vaccine
manufacturing areas.
(d) Control vessels. From the tissue used for the preparation of
cell cultures for growing attenuated mumps virus, an amount of processed
cell suspension equivalent to that used to prepare 500 ml. of cell
culture shall be used to prepare uninfected tissue control materials
which shall be prepared and tested by following the procedures
prescribed in Sec. 630.32(f).
(e) Test samples. Test samples of mumps virus harvests or pools
shall be withdrawn and maintained by following the procedures prescribed
in Sec. 630.32(g).
[38 FR 32068, Nov. 20, 1973, as amended at 50 FR 4138, Jan. 29, 1985]
Sec. 630.53 Reference virus.
An NIH Reference Mumps Virus, Live, shall be obtained from the
Center for Biologics Evaluation and Research
[[Page 108]]
as a control for correlation of virus titers.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
Sec. 630.54 Potency test.
The concentration of live mumps virus shall constitute the measure
of potency. The titration shall be performed in a suitable cell culture
system, free of wild viruses, using either the Reference Mumps Virus,
Live, or a calibrated equivalent strain as a titration control. The
concentration of live mumps virus contained in the vaccine of each lot
under test shall be no less than the equivalent of 5,000 TCID50 of
the reference virus per human dose.
Sec. 630.55 Test for safety.
(a) Tests prior to clarification. Prior to clarification, the
following tests shall be performed on each mumps virus pool prepared in
chick embryo cell culture:
(1) Inoculation of adult mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(1), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(2) Inoculation of suckling mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(2), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(3) Inoculation of monkey cell cultures. A mumps virus pool shall be
tested for adventitious agents in the volume and following the
procedures prescribed in Sec. 630.35(a)(3), and the virus pool is
satisfactory only if equivalent test results are obtained.
(4) Inoculation of other cell cultures. The mumps virus pool shall
be tested for adventitious agents in the volume and following the
procedures prescribed in Sec. 630.35(a)(3), in rhesus or cynomolgus
monkey kidney, in whole chick embryo, and in human cell cultures. In
addition, each virus pool shall be tested in chick embryo kidney in the
same manner except that the volume tested in each cell culture shall be
equivalent to 250 human doses or 25 milliliters, whichever represents a
greater volume. The mumps virus pool is satisfactory only if results
equivalent to those in Sec. 630.35(a)(3) are obtained.
(5) Inoculation of embryonated chicken eggs. A neutralized
suspension of each undiluted mumps virus pool shall be tested in the
volume and following the procedures prescribed in Sec. 630.35(a)(5), and
the virus pool is satisfactory only if there is no evidence of
adventitious agents.
(6) Bacteriological tests. In addition to the tests for sterility
required pursuant to Sec. 610.12 of this chapter, bacteriological tests
shall be performed on each mumps virus pool for the presence of M.
tuberculosis, both avian and human, by appropriate culture methods. The
virus pool is satisfactory only if found negative for M. tuberculosis,
both avian and human.
(7) Test for avian leucosis. If the cultures were not derived from a
certified source and control fluids were not tested for avian leucosis,
the vaccine shall be tested in the volume and following the procedures
prescribed in Sec. 630.35(a)(8). The cultures are satisfactory for
vaccine manufacture if found negative for avian leucosis.
(b) Clarification. The mumps virus fluids shall be clarified by
following the procedures prescribed in Sec. 630.35(c).
[38 FR 32068, Nov. 20, 1973, as amended at 55 FR 47876, Nov. 16, 1990]
Sec. 630.56 General requirements.
(a) Final container tests. In addition to the tests required
pursuant to Sec. 610.14 of this chapter, an immunological and
virological identity test shall be performed on the final container if
it was not performed on each pool or the bulk vaccine prior to filling.
(b) Dose. These standards are based on an individual human
immunizing dose of no less than 5,000 TCID50 of Mumps Virus Vaccine
Live, expressed in terms of the assigned titer of the Reference Mumps
Virus, Live.
(c) Labeling. In addition to the items required by other applicable
labeling provisions of this part, single dose container labeling for
vaccine which is not protected against photochemical deterioration shall
include a statement cautioning against exposure to sunlight.
(d) [Reserved]
[[Page 109]]
(e) Photochemical deterioration; protection. Mumps Virus Vaccine
Live, in multiple dose containers, shall be protected against
photochemical deterioration in accordance with the procedures prescribed
in Sec. 630.36(g).
(f) Samples and protocols. For each lot of vaccine, the following
materials shall be submitted to the Director, Center for Biologics
Evaluation and Research, Food and Drug Administration, 8800 Rockville
Pike, Bethesda, MD 20892:
(1) A protocol which consists of a summary of the history of
manufacture of each lot including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research.
(2) A total of no less than two 25-milliliter volumes, in a frozen
state (-60 deg. C), of preclarification bulk vaccine containing no
preservative, stabilizer, or adjuvant.
(3) A total of no less than 30 containers of the vaccine from each
filling of each bulk lot of single-dose containers. A total of no less
than six 50-dose containers or ten 10-dose containers of the vaccine
from each filling of each bulk lot of multiple-dose containers.
[38 FR 32068, Nov. 20, 1973, as amended at 39 FR 9661, Mar. 13, 1974; 41
FR 10429, Mar. 11, 1976; 49 FR 23834, June 8, 1984; 50 FR 4138, Jan. 29,
1985; 51 FR 15610, Apr. 25, 1986; 55 FR 11013, Mar. 26, 1990]
Subpart G--Rubella Virus Vaccine Live
Sec. 630.60 Rubella Virus Vaccine Live.
(a) Proper name and definition. The proper name of this product
shall be Rubella Virus Vaccine Live, which shall consist of a
preparation of live, attenuated rubella virus.
(b) Criteria for acceptable strains of attenuated rubella virus.
Strains of attenuated rubella virus used in the manufacture of vaccine
shall be identified by (1) historical records including origin and
manipulation during attenuation and (2) antigenic specificity as rubella
virus as demonstrated by tissue culture neutralization tests.
(c) Extraneous agents. Seed virus used for vaccine manufacture shall
be free of all demonstrable extraneous viable microbial agents except
for unavoidable bacteriophage.
(d) Field studies with experimental vaccines. (1) Strains used for
the manufacture of Rubella Virus Vaccine Live, shall have been shown in
field studies with experimental vaccines to be safe and potent in the
group of individuals inoculated, which must include at least 10,000
susceptible individuals. Susceptibility shall be shown by the absence of
neutralizing or hemagglutination-inhibiting antibodies against rubella
virus or by other appropriate methods.
(2) The virus strain used in the field studies shall be propagated
in the same cell culture system that will be used in the manufacture of
the product.
(3) The field studies shall be so conducted that at least 5,000 of
the susceptible individuals must reside when inoculated in areas where
health related statistics are regularly compiled in accordance with
procedures such as those used by the National Center for Health
Statistics. Data in such form as will identify each inoculated person
shall be furnished to the Director, Center for Biologics Evaluation and
Research.
(4) Inoculated persons shall be shown not to be contagious for
contacts through surveillance of rubella susceptible contacts of the
inoculated persons.
(e) Neurovirulence safety test of the virus seed strain in monkeys--
(1) The test. A demonstration shall be made in monkeys of the lack of
neurotropic properties of the seed strain of attenuated rubella virus
used in the manufacture of rubella vaccine. For this purpose and to
establish consistency of manufacture of the vaccine, vaccine from each
of five consecutive lots shall be tested separately in monkeys shown to
be serologically negative for rubella virus antibodies in the following
manner:
(i) A test sample of vaccine removed after clarification but before
final dilution for standardization of virus content shall be used for
the test.
(ii) Vaccine shall be injected by combined intracerebral,
intraspinal, and intramuscular routes into not less than 20 Macaca or
Cercopithecus monkeys or a species found by the Director, Center for
Biologics Evaluation and Research, to be equally suitable for the
purpose. The animals shall be in overt
[[Page 110]]
good health and injected under deep barbiturate anesthesia. The
intramuscular injection shall consist of 1.0 milliliter of test sample
into the right leg muscles. At the same time, 200 milligrams of
cortisone acetate shall be injected into the left leg muscles, and 1.0
milliliter of procaine penicillin (300,000 units) into the right arm
muscles. The intracerebral injection shall consist of 0.5 milliliter of
test sample into each thalamic region of each hemisphere. The
intraspinal injection shall consist of 0.5 milliliter of test sample
into the lumbar spinal cord enlargement.
(iii) The monkeys shall be observed for 17-21 days and symptoms of
paralysis as well as other neurologic disorders shall be recorded.
(iv) At least 90 percent of the test animals must survive the test
period without losing more than 25 percent of their weight except that,
if at least 70 percent of the test animals survive the first 48 hours
after injection, those animals which do not survive this 48-hour test
period may be replaced by an equal number of qualified test animals
which are tested pursuant to paragraphs (e)(1)(i) through (iii) of this
section. At least 80 percent of the injected animals surviving beyond
the first 48 hours must show gross or microscopic evidence of
inoculation trauma in the thalamic area and microscopic evidence of
inoculation trauma in the lumbar region of the spinal cord. If less than
70 percent of the test animals survive the first 48 hours, or if less
than 80 percent of the animals meet the inoculation criteria prescribed
in this paragraph, the test must be repeated.
(v) At the end of the observation period, each surviving animal
shall be autopsied and samples of cerebral cortex and of cervical and
lumbar spinal cord enlargements shall be taken for virus recovery and
identification if needed pursuant to paragraph (e)(1) (vi) of this
section. Histological sections shall be prepared from both spinal cord
enlargements and appropriate sections of the brain and examined.
(vi) Doubtful histopathological findings necessitate (a) examination
of a sample of sections from several regions of the brain in question,
and (b) attempts at virus recovery from the nervous system tissues
previously removed from the animal.
(vii) The lot is satisfactory if the histological and other studies
demonstrate no evidence of changes in the central nervous system
attributable to the presence of unusual neurotropism of the seed virus
or of the presence of extraneous neurotropic agents.
(2) Test results. The rubella virus seed has acceptable
neurovirulence properties for use in vaccine manufacture only if for
each of the five lots: (i) 90 percent of the monkeys survive the
observation period, (ii) the histological and other studies produce no
evidence of changes in the central nervous system attributable to the
presence of unusual neurotropism or replication of the seed virus and
(iii) there is no evidence of the presence of extraneous neurotropic
agents.
(3) Need for additional neurovirulence safety testing. A
neurovirulence safety test as prescribed in this paragraph shall be
performed on vaccine from five consecutive lots whenever a new
production seed lot is introduced or whenever the source of cell culture
substrate must be reestablished and recertified as prescribed in
Sec. 630.62(a), (b) and (d) of this part.
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
49 FR 23834, June 8, 1984; 50 FR 4138, Jan. 29, 1985; 55 FR 11013, Mar.
26, 1990; 55 FR 47876, Nov. 16, 1990]
Sec. 630.61 Clinical trials to qualify for license.
To qualify for license, the antigenicity of Rubella Virus Vaccine
Live, shall be determined by clinical trials, conducted in compliance
with part 56 of this chapter unless exempted under Sec. 56.104 or
granted a waiver under Sec. 56.105, and with part 50 of this chapter,
that follow the procedures prescribed in Sec. 630.31, except that the
immunogenic effect shall be demonstrated by establishing that a
protective antibody response has occurred in at least 90 percent of each
of the five groups of rubella-susceptible individuals, each having
received the parenteral administration of a virus vaccine dose not
greater than that demonstrated to be safe in field studies
[[Page 111]]
when used under comparable conditions.
[46 FR 8956, Jan. 27, 1981, as amended at 50 FR 4138, Jan. 29, 1985]
Sec. 630.62 Production.
(a) Virus cultures. Rubella virus shall be propagated in duck embryo
cell cultures, rabbit renal cultures, or in a cell line found by the
Director, Center for Biologics Evaluation and Research, to meet the
requirements of Sec. 610.18(c) of this chapter.
(b) Virus propagated in duck embryo tissue cell cultures.
Embryonated duck eggs used as a source of duck embryo tissue for the
propagation of rubella virus shall be derived from flocks certified to
be free of avian tuberculosis, the avian leucosis-sarcoma group of
viruses, reticuloendotheliosis virus, and other agents pathogenic for
ducks. Only ducks so certified and in overt good health and which are
maintained in quarantine shall be used as a source of duck embryo tissue
used in the propagation of rubella virus. Ducks in the quarantined flock
that die shall be necropsied and examined for evidence of significant
pathologic lesions. If any such signs or pathologic lesions are
observed, eggs from that flock shall not be used for the manufacture of
Rubella Virus Vaccine Live. Control vessels shall be prepared, observed,
and tested as prescribed in Sec. 630.32(f).
(c) [Reserved]
(d) Virus propagated in rabbit renal tissue cell cultures. Only
rabbits in overt good health which have been maintained in quarantine
individually caged in vermin-proof quarters for a minimum of 6 months,
having had no exposure to other rabbits or animals throughout the
quarantine period, or rabbits born to rabbits while so quarantined,
provided the progency have been kept in the same type of quarantine
continuously from birth shall be used as a source of kidney tissue.
Animals shall be free of antibodies for agents potentially pathogenic
for man unless it has been demonstrated in the license application that
the tests required by Sec. 630.65(c) to be performed on each lot of
vaccine are capable of detecting contamination of agents capable of
producing such antibodies.
(1) Rabbits used for experimental purposes. Rabbits that have been
used previously for experimental or testing purposes with
microbiological agents shall not be used as a source of kidney tissue in
the production of vaccine.
(2) Quarantine and necropsy. Each rabbit shall be examined
periodically during the quarantine period as well as at the time of
necropsy under the direction of a qualified pathologist, physician or
veterinarian having experience with diseases of rabbits, for the
presence of signs or symptoms of ill health, particularly for evidence
of tuberculosis, myxomatosis, fibromatosis, rabbit pox, and other
diseases indigenous to rabbits. If there are any such signs, symptoms or
other significant pathological lesions observed, tissues from that
colony shall not be used in the production of vaccine.
(3) Control vessels. Control vessels shall be prepared, observed and
tested as prescribed in Sec. 630.32(f).
(e) Passage of virus strain in vaccine manufacture. Virus in the
final vaccine shall represent no more than five cell culture passages
beyond the passage used as the seed strain for the manufacture of the
vaccine used to perform the field studies (Sec. 630.60(d)), which
qualified the manufacturer's vaccine strain for license.
(f) Cell cultures in vaccine production areas. Only the cell
cultures used in the propagation of rubella virus vaccine shall be
introduced into rubella virus vaccine production areas.
(g) Test samples. Test samples of rubella virus harvests or pools
shall be withdrawn and maintained by following the procedures prescribed
in Sec. 630.32(g).
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
47 FR 24699, June 8, 1982; 50 FR 4138, Jan. 29, 1985; 55 FR 47876, Nov.
16, 1990]
Sec. 630.63 Reference virus.
A Reference Rubella Virus, Live, shall be obtained from the Center
for Biologics Evaluation and Research as a control for correlation of
virus titers.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
[[Page 112]]
Sec. 630.64 Potency test.
The concentration of live rubella virus shall constitute the measure
of potency. The titration shall be performed in a suitable cell culture
system, using either the Reference Rubella Virus, Live, or a calibrated
equivalent strain as a titration control. The concentration of live
rubella virus contained in the vaccine of each lot under test shall be
no less than the equivalent of 1,000 TCID50 of the reference virus
per human dose.
Sec. 630.65 Test for safety.
(a) Tests prior to clarification of vaccine manufactured in duck
embryo cell cultures. Prior to clarification, the following tests shall
be performed on each rubella virus pool prepared in duck embryo cell
cultures:
(1) Inoculation of adult mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(1), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(2) Inoculation of suckling mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(2), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(3) Inoculation of monkey tissue cell cultures. A rubella virus pool
shall be tested for adventitious agents in the volume and following the
procedures prescribed in Sec. 630.35(a)(3), except that the virus need
not be neutralized by antiserum. The rubella virus pool is satisfactory
only if equivalent test results are obtained.
(4) Inoculation of other cell cultures. The rubella virus pool shall
be tested for adventitious agents in the volume and following the
procedures prescribed in Sec. 630.35(a)(3), in rhesus or cynomolgus
monkey kidney, in chick embryo, duck embryo, and in human cell cultures
except that the virus need not be neutralized by antiserum. The rubella
virus pool is satisfactory only if results equivalent to those in
Sec. 630.35(a)(3) are obtained.
(5) Inoculation of embryonated chicken eggs. A suspension of each
undiluted rubella virus pool shall be tested in the volume and following
the procedures prescribed in Sec. 630.35(a)(5) except that the virus
need not be neutralized by antiserum. The virus pool is satisfactory
only if there is no evidence of adventitious agents.
(6) Inoculation of embryonated duck eggs. A suspension of each
undiluted rubella virus pool shall be tested in embryonated duck eggs,
following the procedures prescribed in Sec. 630.35(a)(5), except that
the virus need not be neutralized by antiserum and the volume of
inoculum per egg shall not exceed 1.0 milliliter. The virus pool is
satisfactory only if there is no evidence of adventitious agents.
(7) Bacteriological tests. In addition to the tests for sterility
required pursuant to Sec. 610.12 of this chapter, bacteriological tests
shall be performed on each rubella virus pool for the presence of M.
tuberculosis, both avian and human, by appropriate culture methods. The
virus pool is satisfactory only if found negative for M. tuberculosis,
both avian and human.
(8) Test for avian leucosis. The vaccine shall be tested for avian
leucosis, in the volume and following the procedures prescribed in
Sec. 630.35(a)(8). The cultures are satisfactory for vaccine manufacture
if found negative for avian leucosis.
(9) Inoculation of cell cultures and embryonated eggs after
neutralization of the virus with antiserum. Each of the tests prescribed
in paragraphs (a)(3), (4), (5), and (6) of this section shall be carried
out also with rubella virus that has been neutralized by the addition of
high titer antiserum of nonhuman, nonsimian and nonavian origin except
that the volume of virus suspension of each undiluted virus pool tested
shall be no less than 5 ml. The rubella antiserum shall have been
prepared by using a rubella virus propagated in a cell culture system
other than that used for the manufacture of the vaccine under test, and
the cell culture system shall be free of extraneous agents which might
elicit antibodies that could inhibit growth of any known extraneous
agents which might be present in the vaccine under test. These tests may
be performed either before or after clarification of the virus. The
virus pool is satisfactory only if the results obtained are
[[Page 113]]
equivalent to those required in those subparagraphs.
(b) [Reserved]
(c) Tests prior to clarification of vaccine manufactured in rabbit
renal cell cultures. Prior to clarification each rubella virus pool
prepared in rabbit renal cell cultures shall be tested as follows:
(1) Inoculation of adult mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(1), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(2) Inoculation of suckling mice. The test shall be performed in the
volume and following the procedures prescribed in Sec. 630.35(a)(2), and
the virus pool is satisfactory only if equivalent test results are
obtained.
(3) Inoculation of monkey tissue cell cultures. A rubella virus pool
shall be tested for adventitious agents in the volume and following the
procedures prescribed in Sec. 630.35(a)(3), except that the virus need
not be neutralized by antiserum. The rubella virus pool is satisfactory
only if equivalent test results are obtained.
(4) Inoculation of other cell cultures. The tests shall be performed
in the volume and following the procedures prescribed in
Sec. 630.35(a)(3) in rhesus or cynomolgus monkey kidney tissue, rabbit
renal tissue and human tissue cell cultures, except that the virus need
not be neutralized by antiserum. The rubella virus pool is satisfactory
only if equivalent test results are obtained.
(5) Inoculation of embryonated chicken eggs. A suspension of each
undiluted rubella virus pool shall be tested in the volume and following
the procedures prescribed in Sec. 630.35(a)(5) except that the virus
need not be neutralized by antiserum. The virus pool is satisfactory
only if there is no evidence of adventitious agents.
(6) Inoculation of rabbits. A minimum of 15 ml. of each virus pool
shall be tested by inoculation into at least five healthy rabbits, each
weighing 1500-2500 grams. Each rabbit shall be injected intradermally in
multiple sites with a total of 1.0 ml. and subcutaneously with 2.0 ml.,
of the virus pool, and the animals observed for at least 30 days. Each
rabbit that dies after the first 24 hours of the test or is sacrificed
because of illness shall be necropsied and the brain and organs removed
and examined. The virus pool is satisfactory only if at least 80 percent
of the rabbits remain healthy and survive the entire period and if all
the rabbits used in the test fail to show lesions of any kind at the
sites of inoculation and fail to show evidence of any viral infection.
(7) Inoculation of guinea pigs. Each of at least five guinea pigs,
each weighing 350-450 grams, shall be inoculated intracerebrally with
0.1 ml. and intraperitoneally with 5 ml. of the undiluted virus pool.
The animals shall be observed for at least 42 days. Each animal that
dies after the first 24 hours of the test or is sacrificed because of
illness, shall be necropsied. All remaining animals shall be sacrificed
and necropsied at the end of the observation period. The virus pool is
satisfactory only if at least 80 percent of all animals remain healthy
and survive the observation period and if all the animals used in the
test fail to show evidence of infection with M. tuberculosis or any
viral infection.
(8) Bacteriological tests. In addition to the tests for sterility
required pursuant to Sec. 610.12 of this chapter, bacteriological tests
shall be performed on each rubella virus pool for the presence of M.
tuberculosis, human, by appropriate culture methods. The rubella virus
pool is satisfactory only if found negative for M. tuberculosis, human.
(9) Tests for adventitious agents. Each virus pool shall be tested
for the presence of such known adventitious agents of rabbits as
toxoplasma, encephalitozoon, herpes cuniculi, the vacuolating virus of
rabbits, rabbit syncytial virus, myxoviruses and reoviruses. The virus
pool is satisfactory only if the results of all tests show no evidence
of any extraneous agent attributable to the rabbit renal tissue or the
vaccine.
(10) Inoculation of cell cultures and embryonated eggs after
neutralization of the virus with antiserum. Each of the tests prescribed
in paragraphs (c)(3), (4), and (5) of this section shall be carried out
also with rubella virus that has been neutralized by the addition of
[[Page 114]]
high titer antiserum of nonhuman, nonsimian and nonrabbit origin
following the procedures and in the volume prescribed in paragraph
(a)(9) of this section. The virus pool is satisfactory only if the
results obtained are equivalent to those required by that paragraph.
(d) Clarification. The rubella virus fluids shall be clarified by
following the procedures prescribed in Sec. 630.35(c).
[38 FR 32068, Nov. 20, 1973, as amended at 40 FR 11719, Mar. 13, 1975;
40 FR 25813, June 19, 1975]
Sec. 630.66 General requirements.
(a) Final container tests. In addition to the tests required
pursuant to Sec. 610.14 of this chapter, an immunological and
virological identity test shall be performed on the final container if
it was not performed on each pool or on the bulk vaccine prior to
filling.
(b) Dose. These standards are based on an individual human
immunizing dose of no less than 1,000 TCID50 of Rubella Virus
Vaccine Live, expressed in terms of the assigned titer of the Reference
Rubella Virus, Live.
(c) Labeling. In addition to the items required by other applicable
labeling provisions of this subchapter, single dose container labeling
for vaccine which is not protected against photochemical deterioration
shall include a statement cautioning against exposure to light.
(d) Photochemical deterioration; protection. Rubella Virus Vaccine
Live, in multiple dose containers, shall be protected against
photochemical deterioration in accordance with the procedures prescribed
in Sec. 630.36(g).
(e) Samples; protocols; offical release. The following shall be
submitted to the Director, Center for Biologics Evaluation and Research,
Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892:
(1) For each lot of vaccine:
(i) A protocol, which consists of a summary of the history of the
manufacture of the lot, including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research.
(ii) A total of no less than two 25-milliliter volumes, in a frozen
state (-60 deg. C.), of preclarification bulk vaccine containing no
preservative or adjuvant.
(iii) A total of no less than 30 containers of the vaccine from each
filling of each bulk lot of single-dose containers. A total of no less
than six 50-dose containers or ten 10-dose containers of the vaccine
from each filling of each bulk lot of multiple-dose containers.
(2) In addition to the requirements of paragraph (e)(1) of this
section, whenever a new production seed lot is introduced, or whenever
the source of cell culture substrate must be reestablished and
recertified, samples consisting of no less than 100 milliliters in 10-
milliliter volumes, in a frozen state (-60 deg. C.), of
postclarification bulk vaccine containing stabilizer but no preservative
or adjuvant, taken from each of 5 consecutive lots of the bulk vaccine.
(3) The product shall not be issued by the manufacturer until
written notification of official release of the lot is received from the
Director, Center for Biologics Evaluation and Research.
[38 FR 32068, Nov. 20, 1973, as amended at 41 FR 10430, Mar. 11, 1976;
42 FR 27582, May 31, 1977; 49 FR 23834, June 8, 1984; 50 FR 4138, Jan.
29, 1985; 51 FR 15610, Apr. 25, 1986; 55 FR 11013, Mar. 26, 1990]
Subpart H--Smallpox Vaccine
Sec. 630.70 Smallpox Vaccine.
(a) Proper name and definition. The proper name of this product
shall be Smallpox Vaccine, which shall be a preparation of live vaccinia
virus obtained from inoculated calves or chicken embryos.
(b) Strains of virus. The strain of seed virus used in the
manufacture of Smallpox Vaccine shall be identified by historical
records including origin and manipulation, and shall meet the sterility
test requirements when tested by the procedure prescribed in Sec. 610.12
of this chapter. The strain of seed virus and every third passage shall
be tested by a rabbit scarification procedure and shown to maintain its
original dermatropic properties. The test procedure is available upon
request from the Director, Center for Biologics Evaluation and Research.
Any new strain shall be shown not to produce a
[[Page 115]]
reactivity in man exceeding that produced by the Reference Smallpox
Vaccine.
[38 FR 32068, Nov. 20, 1973, as amended at 41 FR 51010, Nov. 19, 1976;
49 FR 23834, June 8, 1984; 55 FR 11013, Mar. 26, 1990]
Sec. 630.71 Production.
Vaccinia virus used for the manufacture of vaccine shall be obtained
from vesicles on the skin of an inoculated calf or from inoculated
chorioallantoic membranes of chicken embryos, as set forth below:
(a) Virus from calves--(1) Quarantine. Only calves which, prior to
being placed in quarantine have reacted negatively to tuberculin, were
afebrile and free of ectoparasites, and which shall have met all other
applicable quarantine requirements of Sec. 600.11(f)(2)(i) of this
chapter, shall be used for vaccinia virus production. The quarantine
period shall be at least 14 days. During the last 7 days of the
quarantine period daily morning and afternoon rectal temperatures shall
be taken and calves that do not remain afebrile during that period shall
not be used for virus production.
(2) Inoculation. A larger area of the calf than will be used for
production purposes shall be prepared in a manner comparable to that
appropriate for aseptic surgery, except that the area to be inoculated
must be washed free of all antiseptics that may have a deleterious
effect on virus propagation. The instrument and method used for
scarification must produce a uniform penetration into the epidermis but
must not extend through into the corium.
(3) Incubation. The inoculated calf shall remain in the incubation
room confined to its stall and daily morning and afternoon rectal
temperatures shall be taken to determine that only the expected febrile
condition occurs. If any signs of disease other than vesiculation at the
inoculation site occur, the virus from that calf shall not be used for
vaccine manufacture.
(4) Harvesting. Before harvesting, the calf shall be anesthetized
and killed by exsanguination. Prior to harvesting, the inoculated area
shall be thoroughly cleansed by aseptic techniques. Only the vesicular
material shall be harvested.
(5) Necropsy. A necropsy shall be made of each production calf. The
harvested material shall not be used from any animal suspected of having
an infection other than vaccinia.
(b) Virus from embryonated chicken eggs--(1) Eggs for production.
Embryonated chicken eggs used for propagation of vaccinia virus shall be
derived from flocks found to be free of, and continuously monitored for
freedom from Salmonella pullorum, Mycoplasma species, avian
tuberculosis, fowl pox, Newcastle disease virus, Rous sarcoma virus,
avian leucosis complex of viruses, and other agents pathogenic for
chickens, or appropriate tests shall be performed to demonstrate freedom
of the vaccine from such agents.
(2) Harvesting. Aseptic techniques shall be used in harvesting the
chorioallantoic membranes exhibiting vesicles characteristic of vaccinia
infection.
Sec. 630.72 Reference vaccine.
Reference Smallpox Vaccine and reconstitution fluid shall be
obtained from the Center for Biologics Evaluation and Research and shall
be used in all tests for determining the potency of Smallpox Vaccine.
[38 FR 32068, Nov. 20, 1973, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
Sec. 630.73 Potency test.
Each filling of Smallpox Vaccine shall be tested for potency by the
``pock count'' method as follows:
(a) [Reserved]
(b) Pock counting in embryonated chicken eggs--(1) Dilutions shall
be made starting with no less than 0.5 ml. of the test vaccine and of
the reference vaccine. The same diluent shall be used for all dilutions
of both vaccines. The sample of vaccine in capillary tubes shall be
obtained by pooling the contents of no less than 50 capillaries into a
sterile vessel.
(2) Inoculation of embryonated chicken eggs. One-tenth milliliter of
each dilution of test vaccine shall be inoculated onto the
chorioallantoic membrane of each of at least five embryonated chicken
eggs. The reference vaccine shall be tested in the same manner.
[[Page 116]]
After inoculation, all eggs shall be incubated at
37 deg.Cplus-minus1 deg.C for 48 hours.
(3) Estimation of potency. Only membranes from living embryos shall
be removed and the number of specific lesions thereon shall be counted
and recorded. The number of pock forming units in 1.0 ml. of vaccine
shall be calculated from the number of lesions, the dilution factor and
the volume used, to determine the titer of the undiluted vaccine. The
accuracy of the titration shall be confirmed in each test by performing
simultaneously the same type of titration with the reference vaccine
which shall demonstrate its assigned titer.
(4) Potency requirements--(i) Vaccine intended for multiple pressure
administration. Vaccine intended for multiple pressure administration
shall have a titer at least equivalent to the reference vaccine.
(ii) Vaccine intended for jet injection. Vaccine intended for
administration by jet injector shall have a number of pock forming units
in one human dose at least equivalent to that contained in 0.1 ml. of
the reference vaccine diluted 1:30.
(iii) Heated liquid vaccine. Samples of liquid vaccine from final
containers taken at random shall be incubated at 35 deg. to 37 deg. C.
for at least 18 hours, after which the heated sample shall be tested in
parallel with a sample of unheated vaccine of the same lot, as
prescribed in this paragraph. The vaccine is satisfactory if the heated
sample retains at least one tenth of the potency of the unheated sample.
(iv) Heated dried vaccine. Samples of dried vaccine from final
containers taken at random shall be incubated at 35 deg. to 37 deg. C.
for 30 days, after which the heated sample shall be tested in parallel
with a sample of unheated vaccine of the same lot, as prescribed in this
paragraph. The vaccine is satisfactory if the heated sample retains at
least one-tenth of the potency of the unheated sample.
[38 FR 32068, Nov. 20, 1973, as amended at 41 FR 51010, Nov. 19, 1976]
Sec. 630.74 Tests for safety.
(a) Anaerobes. A 10-milliliter sample representative of the
homogenized viral harvest or pool of several viral harvests shall be
tested for the presence of anaerobes in the following manner: Before the
addition of preservatives other than glycerin, the test sample shall be
inoculated into freshly heated Fluid Thioglycollate Medium using a ratio
of inoculum to culture medium sufficient for optimal bacterial growth.
The test vessels shall be incubated at 35 deg. to 37 deg. C and observed
daily for 10 days for evidence of bacterial growth. If bacterial growth
is observed, the organism(s) shall be identified as to genus. Within 24
to 48 hours of an indication that there may be anaerobic growth, 1.0-
milliliter samples from each vessel showing growth shall be inoculated
subcutaneously into each of at least three mice weighing not more than
20 grams each, and into each of three guinea pigs weighing not more than
350 grams each. The animals shall be observed daily for 6 days for signs
of tetanus or presence of other anaerobes. If the animals show no signs
of tetanus or presence of other anaerobes, additional groups of the same
types and numbers of animals shall be injected 9 days after evidence of
anaerobic bacterial growth is observed in the original planting with
1.0-milliliter samples from each test vessel showing growth. The animals
shall be observed daily for 6 days for signs of tetanus or presence of
other anaerobes. If any animals die within 3 days without having shown
signs of tetanus or presence of other anaerobes, the test shall be
repeated within 18 hours of the deaths, with 0.1-milliliter samples of
the culture from which that animal was inoculated. Samples from the
culture shall be injected into each of three additional test animals of
the same species, and the animals shall be observed daily for 6 days. If
there is any evidence of the presence of pathogenic anaerobes, the viral
harvest may not be used in the manufacture of Smallpox Vaccine.
(b) [Reserved]
(c) Coliform organisms. A 5.0 ml. sample of bulk vaccine shall be
tested for the presence of coliform organisms by the method published by
the American Public Health Association, Inc., in ``Standard Methods for
the Examination of Water and Wastewater'' (13th edition, 1971), section
entitled ``Multiple-Tube Fermentation Technic for
[[Page 117]]
Members of the Coliform Group,'' pages 662-678 and any amendments or
revisions thereof, which section is hereby incorporated by reference and
deemed published herein. Said publication is available at most medical
and public libraries and copies of the pertinent section will be
provided to any manufacturer affected by the provisions of this part
upon request to the Director, Center for Biologics Evaluation and
Research, or to the appropriate Information Center Officer listed in 45
CFR part 5. In addition, an official historic file of the material
incorporated by reference is maintained in the Office of the Director,
Center for Biologics Evaluation and Research, or available for
inspection at the Office of the Federal Register, 800 North Capitol
Street NW., suite 700, Washington, DC 20408. A method different than
that contained in the above cited section may be used to test for the
presence of coliform organisms upon a showing that it is of equal or
greater sensitivity. The ratio of the volume of inoculum to the volume
of culture medium shall be such as will dilute the preservative to a
level that does not inhibit growth of contaminating organisms. The
vaccine is satisfactory if there is no evidence of coliform organisms.
(d) Hemolytic streptococci and coagulase-positive staphylococci.
Each of three 1.0 ml. samples of bulk vaccine shall be spread uniformly
on the surface of separate blood agar plates. The plates shall be
incubated for 48 hours at 35 deg. to 37 deg. C. The vaccine is
satisfactory if there is no evidence of the presence of either hemolytic
streptococci or coagulase-positive staphylococci.
(e) Viable bacteria--(1) Vaccine intended for multiple pressure
administration. Samples of each lot of both bulk and final container
vaccine shall be tested for viable bacteria by a procedure designed to
detect both aerobic and anaerobic growth through a period of 7 days. At
least three 1.0 ml. samples of bulk vaccine and three 0.2 ml. samples of
vaccine derived from not less than three final containers or dilutions
thereof shall be inoculated into a volume of culture medium sufficient
for optimal bacterial growth. The vaccine is satisfactory if it contains
no more than 200 viable organisms per ml.
(2) Vaccine intended for jet injection. Samples of each lot of both
bulk and final container vaccine shall be tested for viable bacteria in
Fluid Thioglycollate Medium prepared in accordance with
Sec. 610.12(e)(1)(i) of this chapter for at least a 7-day test period. A
sample of at least 10.0 ml. of bulk vaccine and 1.0 ml. from each of at
least 20 final containers shall be tested. The ratio of the volume of
the inoculum to the volume of culture medium shall be such as will
dilute the preservative in the inoculum to a level that does not inhibit
growth of contaminating micro-organisms. The vaccine is satisfactory if
it contains no more than one organism per 100 doses of vaccine.
(f) Sterile vaccine. The tests prescribed in paragraphs (c), (d),
and (e) of this section need not be performed on a lot of Smallpox
Vaccine that meets the sterility requirements prescribed in Sec. 610.12
of this chapter.
[38 FR 32068, Nov. 20, 1973, as amended at 41 FR 51010, Nov. 19, 1976;
47 FR 9397, Mar. 5, 1982; 49 FR 23834, June 8, 1984; 55 FR 11013, Mar.
26, 1990]
Sec. 630.75 General requirements.
(a) General safety. Each lot of vaccine shall be tested for safety
as prescribed in Sec. 610.11 of this chapter and shall meet the safety
requirements of that section, except that for liquid Smallpox Vaccine
distributed in capillaries, the test may be performed with a sample of
bulk vaccine taken at the time of filling into final containers.
(b) Preservative. A preservative that meets the requirements of
Sec. 610.15 of this chapter may be used, provided that if the
preservative is phenol, its concentration shall not exceed 0.5 percent.
(c) Labeling. In addition to complying with all other applicable
labeling provisions of this subchapter the package label shall bear the
following:
(1) Vaccine intended for jet injection. (i) A conspicuous statement
that the vaccine is intended for administration by jet injector.
(ii) A statement that the vaccine has been shown by appropriate test
methods to contain not more than one organism per 100 doses or reference
to an enclosed circular that contains such
[[Page 118]]
information, except that such a statement is not required for vaccine
which meets the sterility requirements of Sec. 610.12 of this chapter.
(2) Vaccine intended for multiple pressure administration. A
statement that the vaccine has been shown by appropriate test methods to
contain not more than 200 organisms per ml. or reference to an enclosed
circular that contains such information, except that such a statement is
not required for vaccine which meets the sterility requirements of
Sec. 610.12 of this chapter.
(d) Samples; protocols; official release. (1) For each lot of
vaccine the following shall be submitted to the Director, Center for
Biologics Evaluation and Research, Food and Drug Administration, 8800
Rockville Pike, Bethesda, MD 20892:
(i) A protocol which consists of a summary of the history of
manufacture of each filling including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research.
(ii) Three hundred capillaries from the first filling of a lot of
liquid vaccine, and 200 capillaries from each subsequent filling.
(iii) Two 10 ml. samples of bulk liquid vaccine to be submitted
along with the capillaries from the first filling and taken from the
same vessel from which such capillaries were filled.
(iv) For vaccine intended for jet gun injection, a sample from each
drying consisting of no less than eight 100-dose vials or eight 500-dose
vials of vaccine in final labeled containers, plus sufficient diluent in
final labeled containers to reconstitute the vaccine.
(v) For vaccine intended for multiple pressure administration, a
sample from each drying consisting of no less than eighty 10-dose vials,
ninety 25-dose vials, or eighty 100-dose vials of vaccine in final
labeled containers, plus sufficient diluent in final labeled containers
to reconstitute the vaccine.
(2) The product shall not be issued by the manufacturer until
written notification of official release of the lot is received from the
Director, Center for Biologics Evaluation and Research.
[38 FR 32068, Nov. 20, 1973, as amended at 42 FR 27582, May 31, 1977; 42
FR 56112, Oct. 21, 1977; 49 FR 23834, June 8, 1984; 51 FR 15610, Apr.
25, 1986; 55 FR 11013, Mar. 26, 1990]