[Title 21 CFR 620.4]
[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 620 - ADDITIONAL STANDARDS FOR BACTERIAL PRODUCTS]
[Subpart A - Pertussis Vaccine]
[Sec. 620.4 - Potency test.]
[From the U.S. Government Publishing Office]
21
FOOD AND DRUGS
7
1996-04-01
1996-04-01
false
Potency test.
620.4
Sec. 620.4
FOOD AND DRUGS
FOOD AND DRUG ADMINISTRATION,
BIOLOGICS
ADDITIONAL STANDARDS FOR BACTERIAL PRODUCTS
Pertussis Vaccine
Sec. 620.4 Potency test.
The number of protective units of the total human immunizing dose
shall be estimated for each lot of vaccine from the results of
simultaneous intracerebral mouse protection tests of the vaccine under
test and the U.S. Standard Pertussis Vaccine. The potency test shall be
performed as follows:
(a) Mice. Healthy mice shall be used, all from a single strain and
of the same sex, or an equal number of each sex in each group, with
individual weight varying no more than 4 grams in a single test. In no
event shall any of the mice weigh less than 10 grams or more than 20
grams. A system of randomization shall be used to distribute the mice
into the groups, with respect to shelf position and to determine the
order of challenge. There shall be at least 3 groups consisting of no
less than 16 mice each, for each vaccine. In addition, there shall be at
least 4 groups consisting of no less than 10 mice each, for control
purposes: one group for the challenge dose and 3 groups for titrating
the virulence of the challenge dose.
(b) Vaccination. (1) Five-fold serial dilutions of the vaccine to be
tested and of the standard vaccine shall be made in 0.85 percent sodium
chloride solution. The dilutions of the vaccine under test shall have
the same protective unitage, based on an estimate of 12 units per total
human immunizing dose, as the unitage of the corresponding dilution of
the standard vaccine. Each mouse in each group for vaccination shall be
injected intraperitoneally with 0.5 ml. of the appropriate dilution.
(2) The interval between vaccination and challenge shall be 14 to 17
days. At least 87.5 percent of the mice in each group shall survive the
period between vaccination and challenge and each mouse challenged shall
appear healthy.
(c) The challenge. (1) The challenge culture of Bordetella pertussis
for each test shall be taken from a batch of cultures which have been
maintained by a method, such as freeze-drying, that retains constancy of
virulence.
(2) The challenge and virulence titration doses shall be prepared as
follows: The bacteria shall be harvested from a 20 to 24 hour culture
grown on Bordet-Gengou medium seeded from a rapidly growing culture less
than 48 hours old and uniformly suspended in a solution containing 1.0
percent casein peptone and about 0.6 percent sodium chloride at pH
7.1plus-minus0.1. The suspension, freed from agar particles and
clumps of bacteria, and adjusted to an opacity of 10 units, shall be
diluted in the solution used for suspending the bacteria, to provide in
a volume of 0.03 ml. (i) a challenge dose of 0.0001 opacity units
(1:3000) and (ii) virulence titration doses of \1/50\, \1/250\ and \1/
1250\ respectively of the challenge dose.
(3) Each vaccinated mouse shall be injected intracerebrally with the
challenge dose. The four groups of control mice shall be injected
intracerebrally with the challenge dose and its three dilutions,
respectively. The challenge-dose control mice shall be injected last.
The interval between the removal of the bacteria from the culture medium
and the injection of the last mouse shall not exceed 2\1/2\ hours.
(d) Recording the results. The mice shall be observed for 14 days.
Mice dying within 72 hours after challenge shall be excluded from the
test. Records shall be maintained of the number of mice that die after
72 hours and of the number of mice showing both paralysis and
enlargement of the head at the end of 14 days. All mice that show both
paralysis and enlargement of the head shall be considered as deaths for
the purposes of determining the ED50.
(e) Validity of the test. The test shall be valid provided (1) the
ED50 of the vaccine under test and the standard vaccine is between
the largest and smallest vaccinating doses; (2) the limits of one
standard deviation of each ED50 fall within the range of 64 percent
to 156 percent; (3) the protective response is graded in relation to the
vaccinating doses; (4) the dose-response curves of the vaccine under
test and the standard vaccine are parallel; (5) the challenge dose
contains approximately 200 LD50; (6) the LD50 contains no more
than 300 colony forming units; and (7) the \1/1250\ dilution of the
challenge dose contains no less than 10 and no more than 50 colony
forming units.
(f) Estimate of the potency. The ED50 of each vaccine shall be
calculated by a
[[Page 73]]
method that provides an estimate of the standard deviation. The
protective unit value per total human immunizing dose of the vaccine
under test shall be calculated in terms of the unit value of the
standard vaccine.
(g) Potency requirements. The vaccine shall have a potency of 12
units per total human immunizing dose based upon either a single test
estimate of no less than 8 units or a two-, three- or four-test
geometric mean estimate of no less than 9.6, 10.8, or 12 units,
respectively, except that for the vaccine in a multiple antigen product
containing Poliovirus Vaccine Inactivated, the estimate shall be no less
than 14 units. In no event shall the estimate be more than 36 units.
(h) Test design variation. Variations in the design of the potency
test may be permitted providing the results are demonstrated to be of
equal or greater precision.
[38 FR 32064, Nov. 20, 1973, as amended at 50 FR 4137, Jan. 29, 1985]