EUROPEAN PHARMACOPOEIA 5.

0 Varicella vaccine (live)

Water (2.5.12) : 1.5 per cent to 4.0 per cent, determined Neurovirulence (2.6.18). The working seed lot complies
on the contents of the capsule or of the container by the with the test for neurovirulence of live virus vaccines.
semi-micro determination of water. VIRUS PROPAGATION AND HARVEST
NUMBER OF LIVE BACTERIA All processing of the cell bank and subsequent cell cultures
Carry out the test using not fewer than five dosage units. is done under aseptic conditions in an area where no other
Homogenise the contents of the dosage units in a 9 g/l cells are handled. Approved animal (but not human) serum
solution of sodium chloride R at 4 °C using a mixer in a may be used in the media. Serum and trypsin used in the
cold room with sufficient glass beads to emerge from the preparation of cell suspensions and media are shown to be
liquid. Immediately after homogenisation prepare a suitable free from extraneous agents. The cell culture medium may
dilution of the suspension using cooled diluent and inoculate contain a pH indicator such as phenol red and approved
brain heart infusion agar ; incubate at 36 ± 1 °C for 20 h antibiotics at the lowest effective concentration. It is
to 36 h. The vaccine contains not fewer than 2 × 10 live9 preferable to have a substrate free from antibiotics during
S. typhi Ty 21a bacteria per dosage unit. production. 5 per cent, but not less than 50 ml, of the cell
cultures employed for vaccine production is set aside as
LABELLING uninfected cell cultures (control cells). The infected cells
The label states : constituting a single harvest are washed, released from the
— the minimum number of live bacteria per dosage unit, support surface and pooled. The cell suspension is disrupted
by sonication.
— that the vaccine is for oral use only.
Only a virus harvest that complies with the following
01/2005:0648 requirements may be used in the preparation of the final
bulk vaccine.
VARICELLA VACCINE (LIVE) Identification. The virus harvest contains virus that is
identified as varicella virus by serum neutralisation in cell
Vaccinum varicellae vivum culture, using specific antibodies.
DEFINITION Virus concentration. The concentration of infective virus
in virus harvests is determined as prescribed under Assay
Varicella vaccine (live) is a freeze-dried preparation of a to monitor consistency of production and to determine the
suitable attenuated strain of Herpesvirus varicellae. The dilution to be used for the final bulk vaccine.
vaccine is reconstituted immediately before use, as stated on
the label, to give a clear liquid that may be coloured owing Extraneous agents (2.6.16). Use 50 ml for the test in cell
to the presence of a pH indicator. cultures.
PRODUCTION Control cells. The control cells of the production cell culture
from which the single harvest is derived comply with a test
The production of vaccine is based on a virus seed-lot system for identity and with the requirements for extraneous agents
and a cell-bank system. The production method shall have (2.6.16).
been shown to yield consistently live varicella vaccines of
adequate immunogenicity and safety in man. The virus in FINAL BULK VACCINE
the final vaccine shall not have been passaged in cell cultures Virus harvests that comply with the above tests are pooled
beyond the 38th passage from the original isolated virus. and clarified to remove cells. A suitable stabiliser may be
The production method is validated to demonstrate that the added and the pooled harvests diluted as appropriate.
product, if tested, would comply with the test for abnormal Only a final bulk vaccine that complies with the following
toxicity for immunosera and vaccines for human use (2.6.9). requirements may be used in the preparation of the final lot.
SUBSTRATE FOR VIRUS PROPAGATION Bacterial and fungal contamination. Carry out the test for
The virus is propagated in human diploid cells (5.2.3). sterility (2.6.1) using 10 ml for each medium.
VIRUS SEED LOT FINAL LOT
The strain of varicella virus shall be identified as being The final bulk vaccine is distributed aseptically into sterile,
suitable by historical records which shall include information tamper-proof containers and freeze-dried to a moisture
on the origin of the strain and its subsequent manipulation. content shown to be favourable to the stability of the
The virus shall at no time have been passaged in continuous vaccine. The containers are then closed so as to prevent
cell lines. Seed lots are prepared in the same kind of cells contamination and the introduction of moisture.
as those used for the production of the final vaccine.
To avoid the unnecessary use of monkeys in the test Only a final lot that is satisfactory with respect to each of the
for neurovirulence, virus seed lots are prepared in large requirements given below under Identification, Tests and
quantities and stored at temperatures below − 20 °C, if Assay may be released for use. Provided that the test for
freeze-dried, or below − 60 °C, if not freeze-dried. bovine serum albumin has been carried out with satisfactory
Only a virus seed lot that complies with the following results on the final bulk vaccine, it may be omitted on the
requirements may be used for virus propagation. final lot.
Identification. The master and working seed lots are
identified as varicella virus by serum neutralisation in cell IDENTIFICATION
culture, using specific antibodies. When the vaccine reconstituted as stated on the label is
Virus concentration. The virus concentration of the master mixed with specific Herpesvirus varicellae antibodies, it is
and working seed lots is determined as prescribed under no longer able to infect susceptible cell cultures.
Assay to monitor consistency of production.
Extraneous agents (2.6.16). The working seed lot complies TESTS
with the requirements for seed lots for live virus vaccines ; a Bacterial and fungal contamination. The reconstituted
sample of 50 ml is taken for the test in cell cultures. vaccine complies with the test for sterility (2.6.1).

General Notices (1) apply to all monographs and other texts 709
Yellow fever vaccine (live)
Bovine serum albumin. Not more than 0.5 µg per human
dose, determined by a suitable immunochemical method
(2.7.1).
Water (2.5.12). Not more than 3.0 per cent, determined by
the semi-micro determination of water.
ASSAY
Titrate for infective virus, using at least 10 cell cultures for
each fourfold dilution or by a technique of equal precision.
Use a suitable virus reference preparation to validate each
assay. The virus concentration is not less than the minimum
stated on the label.
LABELLING
The label states :
— the strain of virus used for the preparation of the vaccine,
— the type and origin of the cells used for the preparation
of the vaccine,
— that contact with disinfectants is to be avoided,
— the minimum virus concentration,
— that the vaccine is not to be administered to pregnant
women,
— the time within which the vaccine must be used after
reconstitution.
01/2005:0537
YELLOW FEVER VACCINE (LIVE)
Vaccinum febris flavae vivum
DEFINITION
Yellow fever vaccine (live) is a freeze-dried preparation of
the 17D strain of yellow fever virus grown in fertilised hen
eggs. The vaccine is reconstituted immediately before use, as
stated on the label, to give a clear liquid.
PRODUCTION
The production of vaccine is based on a virus seed-lot
system. The production method shall have been shown to
yield consistently yellow fever vaccine (live) of acceptable
immunogenicity and safety for man.
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)
modified as follows for the test in guinea-pigs : inject ten
human doses into each guinea-pig and observe for 21 days.
Reference preparation. In the test for neurotropism,
a suitable batch of vaccine known to have satisfactory
properties in man is used as the reference preparation.
SUBSTRATE FOR VIRUS PROPAGATION
Virus for the preparation of master and working seed lots
and of all vaccine batches is grown in the tissues of chick
embryos from a flock free from specified pathogens (5.2.2).
SEED LOTS
The 17D strain shall be identified by historical records
that include information on the origin of the strain and its
subsequent manipulation. Virus seed lots are prepared in
large quantities and stored at a temperature below − 60 °C.
Master and working seed lots shall not contain any human
protein or added serum.
Unless otherwise justified and authorised, the virus in the
final vaccine shall be between passage levels 204 and 239
from the original isolate of strain 17D. A working seed lot
shall be only one passage from a master seed lot. A working
seed lot shall be used without intervening passage as the
710 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
inoculum for infecting the tissues used in the production
of a vaccine lot, so that no vaccine virus is more than one
passage from a seed lot that has passed all the safety tests.
Only a virus seed lot that complies with the following
requirements may be used for virus propagation.
Identification. The master and working seed lots are
identified as containing yellow fever virus by serum
neutralisation in cell culture, using specific antibodies.
Extraneous agents (2.6.16). Each working seed lot complies
with the following tests :
Bacterial and fungal sterility
Mycoplasmas
Mycobacteria
Avian viruses
Test in adult mice (intraperitoneal inoculation only)
Test in guinea-pigs
Tests in monkeys. Each master and working seed lot
complies with the following tests in monkeys for viraemia
(viscerotropism), immunogenicity and neurotropism.
The monkeys shall be Macaca sp. susceptible to yellow fever
virus and shall have been shown to be non-immune to yellow
fever at the time of injecting the seed virus. They shall be
healthy and shall not have received previously intracerebral
or intraspinal inoculation. Furthermore, they shall not have
been inoculated by other routes with neurotropic viruses or
with antigens related to yellow fever virus. Not fewer than
ten monkeys are used for each test.
Use a test dose of 0.25 ml containing the equivalent of not
less than 5000 mouse LD50 and not more than 50 000 mouse
LD50, determined by a titration for infectious virus and using
the established equivalence between virus concentration and
mouse LD50 (see under Assay). Inject the test dose into one
frontal lobe of each monkey under anaesthesia and observe
the monkeys for not less than 30 days.
Viraemia (Viscerotropism). Viscerotropism is indicated by
the amount of virus present in serum. Take blood from each
of the test monkeys on the second, fourth and sixth days
after inoculation and prepare serum from each sample.
Prepare 1:10, 1:100 and 1:1000 dilutions from each serum
and inoculate each dilution into a group of at least six cell
culture vessels used for the determination of the virus
concentration. The seed lot complies with the test if none of
the sera contains more than the equivalent of 500 mouse
LD50 in 0.03 ml and at most one serum contains more than
the equivalent of 100 mouse LD50 in 0.03 ml.
Immunogenicity. Take blood from each monkey 30 days
after the injection of the test dose and prepare serum
from each sample. The seed lot complies with the test if
at least 90 per cent of the test monkeys are shown to be
immune, as determined by examining their sera in the test
for neutralisation of yellow fever virus described below.
It has been shown that a low dilution of serum (for example,
1:10) may contain non-specific inhibitors that influence this
test ; such serum shall be treated to remove inhibitors. Mix
dilutions of at least 1:10, 1:40 and 1:160 of serum from
each monkey with an equal volume of 17D vaccine virus
at a dilution that will yield an optimum number of plaques
with the titration method used. Incubate the serum-virus
mixtures in a water-bath at 37 °C for 1 h and then cool in
iced water ; add 0.2 ml of each serum-virus mixture to each of
four cell-culture plates and proceed as for the determination
of virus concentration. Inoculate similarly ten plates with
the same amount of virus plus an equal volume of a 1:10
dilution of monkey serum known to contain no neutralising
antibodies to yellow fever virus. At the end of the observation
period, compare the mean number of plaques in the plates