(Ph Eur monograph 0216)
The label may state 'Rab/Vac'.
Rabies vaccine for human use prepared in cell cultures is a
freeze-dried preparation of a suitable strain of fixed rabies virus grown in cell cultures
and inactivated by a validated method.
The vaccine is reconstituted immediately before use as stated
on the label to give a clear liquid that may
be coloured owing to the presence of a pH indicator.
The production of the vaccine is based on a virus seed-lot
system and, if a cell line is used for virus propagation, a cell-bank system. The
production method shall have been shown to yield consistently vaccines that comply with
the requirements for immunogenicity, safety and stability. Unless otherwise justified and
authorised, the virus in the final vaccine shall not have undergone more passages from the
master seed lot than was used to prepare the vaccine shown in clinical studies to be
satisfactory with respect to safety and efficacy; even with authorised exceptions, the
number of passages beyond the level used for clinical studies shall not exceed five.
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal toxicity for immunosera and
vaccines for human use (2.6.9).
SUBSTRATE FOR VIRUS PROPAGATION
The virus is propagated in a human diploid cell line (5.2.3),
in a continuous cell line approved by the competent authority or in cultures of
chick-embryo cells derived from a flock free from specified pathogens (5.2.2).
The strain of rabies virus used shall be identified by
historical records that include information on the origin of the strain and its subsequent
Working seed lots are prepared by not more than five passages
from the master seed lot.
Only a working seed lot that complies with the following tests
may be used for virus propagation.
Each working seed lot is identified as rabies virus using
The virus concentration of each working seed lot is determined
by a cell culture method using immunofluorescence, to ensure consistency of production.
Extraneous agents (2.6.16)
The working seed lot complies with the requirements for virus
seed lots. If the virus has been passaged in mouse brain, specific tests for murine
viruses are carried out.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures
are done under aseptic conditions in an area where no other cells are handled. Approved
animal (but not human) serum may be used in the media, but the final medium for
maintaining cell growth during virus multiplication does not contain animal serum; the
media may contain human albumin complying with the monograph on Human albumin solution (0255).
Serum and trypsin used in the preparation of cell suspensions and media are shown to be
free from infectious extraneous agents; trypsin complies with the monograph on Trypsin (0694).
The cell culture media may contain a pH indicator such as phenol red and approved
antibiotics at the lowest effective concentration. Not less than 500 ml of the cell
cultures employed for vaccine production are set aside as uninfected cell cultures
(control cells). The virus suspension is harvested on one or more occasions during
incubation. Multiple harvests from the same production cell culture may be pooled and
considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the inactivated viral harvest.
The single harvest contains virus that is identified as rabies
virus using specific antibodies.
Titrate for infective virus in cell cultures; the titre is
used to monitor consistency of production.
The control cells of the production cell culture from which
the single harvest is derived comply with a test for identification and with the
requirements for extraneous agents (2.6.16).
PURIFICATION AND INACTIVATION
The virus harvest may be concentrated and/or purified by
suitable methods; the virus harvest is inactivated by a validated method at a fixed, well
defined stage of the process which may be before, during or after any concentration or
purification. The method shall have been shown to be capable of inactivating rabies virus
without destruction of the immunogenic activity. If betapropiolactone is used, the
concentration shall at no time exceed 1:3500.
Only an inactivated viral suspension that complies with the
following requirements may be used in the preparation of the final bulk vaccine.
Carry out an amplification test for residual infectious rabies
virus immediately after inactivation or using a sample frozen immediately after
inactivation and stored at -70C. Inoculate a quantity of inactivated viral suspension
equivalent to not less than 25 doses of vaccine into cell cultures of the same type as
those used for production of the vaccine. Make a passage after 7 days. Maintain the
cultures for a further 14 days and then examine the cell cultures for rabies virus using
an immunofluorescence test. No rabies virus is detected.
Residual host-cell DNA
If a continuous cell line is used for virus propagation, the
content of residual host-cell DNA, determined using a suitable method as described in Products
of recombinant DNA technology (0784), is not greater than 100 pg per single
FINAL BULK VACCINE
The final bulk vaccine is prepared from one or more
inactivated viral suspensions. An approved stabiliser may be added to maintain the
activity of the product during and after freeze-drying.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
Determine the glycoprotein content by a suitable
immunochemical method (2.7.1), for example, single-radial immunodiffusion, enzyme-linked
immunosorbent assay or an antibody-binding test. The content is within the limits approved
for the particular product.
The final bulk vaccine complies with the test for sterility,
carried out using 10 ml for each medium.
The final bulk vaccine is distributed aseptically into sterile
containers and freeze-dried to a moisture content shown to be favourable to the stability
of the vaccine. The containers are then closed so as to avoid contamination and the
introduction of moisture.
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be released for use. Provided that
the test for inactivation has been carried out with satisfactory results on the
inactivated viral suspension and the test for bovine serum albumin has been carried out
with satisfactory results on the final bulk vaccine, these tests may be omitted on the
The vaccine is shown to contain rabies virus antigen by a
suitable immunochemical method (2.7.1) using specific antibodies, preferably monoclonal;
alternatively, the assay serves also to identify the vaccine.
Inoculate a quantity equivalent to not less than 25 human
doses of vaccine into cell cultures of the same type as those used for production of the
vaccine. Make a passage after 7 days. Maintain the cultures for a further 14 days and then
examine the cell cultures for rabies virus using an immunofluorescence test. No rabies
virus is detected.
Bovine serum albumin
Not more than 50 ng per single human dose, determined by a
suitable immunochemical method (2.7.1).
The vaccine complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than 25 IU per single human dose.
The vaccine complies with the test for pyrogens. Unless
otherwise justified and authorised, inject into each rabbit a single human dose of the
vaccine diluted to ten times its volume.
Not more than 3.0 per cent, determined by the semi-micro
determination of water.
The potency of rabies vaccine is determined by comparing the
dose necessary to protect mice against the effects of a lethal dose of rabies virus,
administered intracerebrally, with the quantity of a reference preparation of rabies
vaccine necessary to provide the same protection. For this comparison a reference
preparation of rabies vaccine, calibrated in International Units, and a suitable
preparation of rabies virus for use as the challenge preparation are necessary.
The International Unit is the activity contained in a stated
quantity of the International Standard. The equivalence in International Units of the
International Standard is stated by the World Health Organisation.
The test described below uses a parallel-line model with at
least three points for the vaccine to be examined and the reference preparation. Once the
analyst has experience with the method for a given vaccine, it is possible to carry out a
simplified test using a single dilution of the vaccine to be examined. Such a test enables
the analyst to determine that the vaccine has a potency significantly higher than the
required minimum but will not give full information on the validity of each individual
potency determination. The use of a single dilution allows a considerable reduction in the
number of animals required for the test and must be considered by each laboratory in
accordance with the provisions of the European Convention for the Protection of Vertebrate
Animals used for Experimental and other Scientific Purposes.
Selection and distribution of the test animals
Use in the test healthy female mice about 4 weeks old, each
weighing 11 g to 15 g, and from the same stock. Distribute the mice into six groups of a
size suitable to meet the requirements for validity of the test and, for titration of the
challenge suspension, four groups of five.
Preparation of the challenge suspension
Inoculate mice intracerebrally with the CVS strain of rabies
virus and when the mice show signs of rabies, but before they die, sacrifice them, remove
the brains and prepare a homogenate of the brain tissue in a suitable diluent. Separate
gross particulate matter by centrifugation and use the supernatant liquid as the challenge
suspension. Distribute the suspension in small volumes in ampoules, seal and store at a
temperature below -60C. Thaw one ampoule of the suspension and make serial dilutions in
a suitable diluent. Allocate each dilution to a group of five mice and inject
intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe
the mice for 14 days. Calculate the LD50 of the undiluted suspension using the
number in each group that, between the fifth and fourteenth days, die or develop signs of
Determination of potency of the vaccine to be
Prepare three five-fold serial dilutions of the vaccine to be
examined and three fivefold serial dilutions of the reference preparation. Prepare the
dilutions such that the most concentrated suspensions may be expected to protect more than
50 per cent of the animals to which they are administered and the least concentrated
suspensions may be expected to protect less than 50 per cent of the animals to which they
are administered. Allocate the six dilutions one to each of the six groups of mice and
inject intraperitoneally into each mouse 0.5 ml of the dilution allocated to its group.
After 7 days, prepare three identical dilutions of the vaccine to be examined and of the
reference preparation and repeat the injections. Seven days after the second injection,
prepare a suspension of the challenge virus such that, on the basis of the preliminary
titration, 0.03 ml contains about 50 LD50. Inject intracerebrally into each
vaccinated mouse 0.03 ml of this suspension. Prepare three suitable serial dilutions of
the challenge suspension. Allocate the challenge suspension and the three dilutions one to
each of the four groups of five control mice and inject intracerebrally into each mouse
0.03 ml of the suspension or one of the dilutions allocated to its group. Observe the
animals in each group for 14 days and record the number in each group that die or show
signs of rabies in the period 5 days to 14 days after challenge.
The test is not valid unless: for both the vaccine to be
examined and the reference preparation the 50 per cent protective dose lies between the
largest and smallest doses given to the mice; the titration of the challenge suspension
shows that 0.03 ml of the suspension contained not less than 10 LD50; the
statistical analysis shows a significant slope and no significant deviations from
linearity or parallelism of the dose-response lines; the fiducial limits of error (P
= 0.95) are not less than 25 per cent and not more than 400 per cent of the estimated
The vaccine complies with the test if the estimated potency is
not less than 2.5 IU per human dose.
The label states the biological origin of the cells used for
the preparation of the vaccine.