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GENERAL REQUIREMENTS FOR THE PRODUCTION

AND CONTROL OF INACTIVATED MAMMALIAN
BACTERIAL AND VIRAL VACCINES FOR
VETERINARY USE

Guideline Title

Legislative Basis
Date of First Adoption
Date of Entry into Force
Status
Previous Titles
Other References
Additional Notes

General Requirements for the Production and Control of

Inactivated Mammalian Bacterial and Viral Vaccines for
Veterinary Use
Directive 81/852/EEC as amended
March 1992
September 1992
Last revised March 1992
General Requirements for Inactivated Mammalian
Vaccines (GRIMV)
III/3181/91
This note for guidance is intended to provide general
guidance on the type of data which should be included i n
applications for marketing authorisations for inactivated
mammalian bacterial and viral vaccines. It is intended to
supplement Directive 81/852/EEC as amended, and should
be read in conjunction with that Directive.

CONTENTS
DEFINITIONS AND GENERAL REQUIREMENTS
1.

STARTING MATERIALS

2.

FINISHED PRODUCT - ASSAY RESULTS REQUIRED IN THE APPLICATION FOR

MARKETING AUTHORISATION

3.

FINISHED PRODUCT - BATCH TESTING

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GENERAL REQUIREMENTS FOR THE PRODUCTION

AND CONTROL OF INACTIVATED MAMMALIAN
BACTERIAL AND VIRAL VACCINES FOR
VETERINARY USE

This document is intended to provide general guidance on the type of data which should be
included in applications for marketing authorisations. The guidelines are intended to
supplement Directive 81/852/EEC, as amended by Directive 92/18/EEC, and must be read i n
conjunction with that Directive.

DEFINITIONS AND GENERAL REQUIREMENTS

DEFINITIONS
Master Seed (MS) a collection of aliquots of a preparation, for use in the preparation of
testing of a product, distributed into containers in a single operation and processed together
in such a manner as to ensure uniformity, and processed and stored in such a manner as to
ensure stability.
Master Cell Seed (MCS) a collection of aliquots of a preparation of cells, for use in the
preparation of a product, distributed into containers in a single operation and processed
together in such a manner as to ensure uniformity, and processed and stored in such a
manner as to ensure stability.
Seed Lot System a system according to which successive batches of product are prepared
using the same Master Cell Seed or Master Seed.
Working Seed Lot a collection of aliquots of a preparation consisting of a passage level
between MS and the last passage, which forms the finished product, for use in the preparation
of finished product, distributed into containers in a single operation and processed together
in such a manner as to ensure uniformity, and processed and stored in such a manner as to
ensure stability.
Working Cell Seed (WCS) a collection of aliquots of a preparation of cells, for use in the
preparation and testing of a product, consisting of cells of a passage level intermediate
between Master Cell Seed and those used for production, distributed into containers in a
single operation and processed together in such a manner as to ensure uniformity, and
processed and stored in such a manner as the ensure stability.
Primary Cell Cultures cultures of cells, essentially unchanged from those in the animal
tissues from which they have been prepared and being no more than 5 in vitro passages to
production level from the initial preparation from the animal tissue.
Batch a defined quantity of starting material, packaging material or product processed i n
one process or series of processes so that it can be expected to be homogenous.

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To complete certain stages of manufacture, it may be necessary to divide a batch into a

number of subbatches, which are further processed in one process or series of processes, so
that each subbatch can be expected to be homogenous.
GENERAL REQUIREMENTS
All inactivated vaccines shall normally comply with these general guidelines unless
species specific guidelines indicate differently.
Additional requirements with regard to specific contaminating agents (e.g. BSE) laid down
in other documents should also be complied with.
Compliance with the guidelines provides as assurance that the research and development
work undertaken will be considered valid by all the Member States. Nevertheless, in order
not to place undue constraints on scientific research, an alternative approach to the one
described in a guideline may be used, if it can be shown that this is justified.
Preference should always be given to tests and methods described in the European
Pharmacopoeia, or failing this, in the pharmacopoeia of a Member State. If other tests and
methods are used, proof must be supplied that they allow to meet the quality requirements of
that pharmacopoeia.

1.

STARTING MATERIALS

1.1

Substances of animal origin

Substances of animal origin (e.g. serum, trypsin and serum albumin) may be used during
the manufacture of veterinary immunological products, as ingredients of culture media etc.
or as added constituents of vaccines or diluents. Wherever practicable, manufacturers are
encouraged to minimise the use of such substances.
Certain restrictions are placed upon the use of these substances in this way in order to
minimise the risk associated with pathogens which may be present in these materials. These
restrictions are not placed on substances sterilised by a suitable validated method.
The use of substances of animal origin as constituents of vaccines or diluents is not
generally acceptable except where such substances are sterilised by a suitable validated
method. Where the use of such substances has been shown to be essential and sterilisation i s
not possible, the requirements described in paragraphs 1.1.1 to 1.1.4 shall apply.
Substances of animal origin used during production should be either sterilised or subject to
an inactivation procedure by a suitable validated method or tested for the absence of
extraneous organisms in accordance with paragraphs 1.1.1 1.1.4.
In addition to the restrictions described below, manufacturers may need to comply with
restrictions, which may be imposed on the handling of substances of animal origin in the
vaccine manufacturing premises.
The restrictions imposed by these sections may need to be varied in accordance with changes
in the disease situations in the country of origin and in Europe.

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1.1.1 Source
This risk related to the animal diseases occurring in the proposed country of origin of the
substance and to the potential of infectious diseases occurring in the source species, i n
relation with the proposed recipient species should be carefully evaluated. The strictest
possible selection criteria should be applied, in particular for substances for use in products
for the same species and for substances of porcine, bovine, caprine and ovine origin.

1.1.2 Preparation
Substances of animal origin shall be prepared from a homogeneous bulk, designated with a
batch number. A batch may contain substances derived from as many animals as is desired
but once designated and given a batch number, a batch shall not be added to or contaminated
in any way.
The batch
of animal
procedure
substance

test protocol shall contain the batch number and country of origin of all substances
origin used. Where applicable, it shall also contain details of the inactivation
to which the substance has been subjected and details of tests performed on the
and results obtained.

All batches of substances shall be shown to be free from contaminants as described below
and/or shall be subject to a suitable inactivation procedure. It is the responsibility of the
manufacturer to decide whether the testing carried out by the supplier is sufficient to meet the
requirements.

1.1.3 Inactivation
The inactivation procedure chosen shall have been shown to be capable of reducing the titre,
in the substance concerned, of certain potential contaminants by at least 106. If this titre
reduction cannot be experimentally demonstrated, then kinetic studies for the inactivation
procedure must be carried out and shown satisfactory, taking into account the possible level
of initial contamination.
The list of potential contaminating organisms that the procedure should be shown to be
capable of inactivating should be appropriate to the particular species of origin of the
substance. The evidence for the efficacy of the procedure, which must relate to the current
circumstances, may take the form of references to published literature or experimental
evidence generated by the manufacturer.
The process used during manufacture of the product for inactivation of the active ingredients
may be suitable for inactivation of other substances, but this must be demonstrated.

1.1.4 Tests
For examination of the substance for freedom from contaminants, any solid substance
should be dissolved/suspended in a suitable medium in such a way as to create a
solution/suspension that is at least 30% substances (w/v). If the substance is not soluble or
when cytotoxic reactions occur, a lower concentration may be used.
1.1.4.1

Freedom from extraneous viruses

The solution/suspension of the solid substance, or undiluted liquid substance should be tested
for contaminants by suitably sensitive methods. The methods employed should include
testing in suitably sensitive cell cultures including primary cells from the same species as
the test substance. A proportion of the cells should be passaged at least twice. The cells should
be observed regularly for 21 days for cytopathic effects. At the end of each 7-day period during

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this time, a proportion of the original cultures should be fixed, stained and examined for
cytopathic effects and a proportion tested for haemadsorbing agents. A proportion should also
be tested for specific agents by appropriate serodiagnostic tests.
In case of viral contamination, the batch should be discarded.
1.1.4.2

Sterility and mycoplasma

Before use, substances shall be tested for sterility or properly sterilised to eliminate any
bacterial, fungal or mycoplasma contaminants.
Any batch of substance that is found to contain living organisms of any kind is not
satisfactory and must be discarded. Alternatively, one reprocessing and retesting may be
carried out. If the batch is still found to be contaminated, it must be discarded. If the batch i s
considered to be free from contamination after this reprocessing then the material may be
used but an explanation should be submitted with the batch test protocol giving the reasons for
the initial failure.

1.2 Cell substrates

1.2.1 General requirements
If a virus can be efficaciously grown on cell cultures based on a seed lot system of
established cell lines, no mammalian primary cells should be used.
Permanently infected cells shall comply with the appropriate requirements described below.
The cells must be shown to be infected only with the agent stated.

1.2.2 Requirements for cell lines

Cell substrates used in manufacture shall normally be produced according to a Seed Lot
System. Each MCS shall be assigned a specific code for identification purposes. The MCS
shall be stored in aliquots at 70 C or lower. Production of vaccine shall not normally be
undertaken on cells further than 20 passages from the MCS.
Where suspension cultures are used, an increase in cells numbers equivalent
approximately three population doublings should be considered equivalent to one passage.

to

If cells beyond this passage level are to be used for production, the applicant should
demonstrate, by validation or further testing, that the production cells are essentially
similar to the MCS with regard to their biological characteristics and purity and that use of
such cells has no deleterious effect on vaccine production.
The history of the cell line must be known in detail and recorded in writing (e.g. origin,
number of passages and media used for their multiplication, storage conditions).
The manufacturer must describe the method of preserving and using the cells, including
details of how it is ensured that the maximum number of passages permitted is not exceeded
during product manufacture. A sufficient number of MCS and WCS cells must be kept
available for testing by the licensing authorities.
The checks described below should be carried out on a culture of the MCS and WCS or on
cells from the WCS at the highest passage level used for production (see Table 1) and derived
from a homogeneous representative sample. The representative nature of this sample must be
proven.

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Table 1: Stages of cell culture at which testing shall be carried out

general microscopy
bacteria/fungi
mycoplasma
viruses
identification of species
karyology
1.2.2.1

MCS
+
+
+
+
+
+

WCS
+
+
+
+
-

Cells from WCS at highest passage level

+
+
+

Extraneous contaminants

1.2.2.1.1 General
The cells must be checked for their appearance under the microscope, for their rate of growth
and for other factors which will provide information on the state of health of the cells.
1.2.2.1.2 Bacteria and fungi
The cells must be checked for contamination with bacteria or fungi. Contaminated cells
must be discarded.
1.2.2.1.3 Mycoplasma
The cells must be checked for freedom from mycoplasma and pass the test for freedom from
mycoplasma.
1.2.2.1.4 Viruses
The cells must not be contaminated by viruses and the checks must be performed in the
following manner:
The monolayers tested must be at least 70 cm 2 , prepared and maintained using a medium
and additives, and grown under similar conditions to those used for the preparation of the
biological product.
The monolayers must be maintained in culture for a total of at least 28 days. Subcultures
should be made at 7-day intervals, unless the cells do not survive for this length of time,
when the subcultures should be made on the latest day possible. Sufficient cells, in suitable
containers, must be produced for the final subculture to carry out the tests specified below.
The monolayers must be examined regularly throughout the incubation period for the
possible presence of cytopathic effects (cpe) and at the end of the observation period for cpe,
haemadsorbent viruses, and specific viruses by immunofluorescence and other appropriate
tests as indicated below.
1.2.2.1.4.1

Detection of cytopathic viruses

Two monolayers of at least 6 cm2 each must be stained with an appropriate cytological stain.
Examine the entire area of each stained monolayer for any inclusion bodies, abnormal
numbers of giant cells or any other lesion indicative of a cellular abnormality which might
be attributable to a contaminant.
1.2.2.1.4.2

Detection of haemadsorbent viruses

Monolayers totalling at least 70 cm2 must be washed several times with an appropriate buffer
and a sufficient volume of a suspension of appropriate red blood cells added to cover the

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surface of the monolayer evenly. After different incubation times examine cells for the
presence of haemadsorption.
1.2.2.1.4.3

Detection of specified viruses

Tests should be carried out for freedom of contaminants specific for the species of origin of
the cell line and for the species for which the product is intended.
Sufficient cells on appropriate supports must be prepared to carry out tests for the agents
specified. Appropriate positive controls must be included in each test. The cells are subjected
to appropriate tests using fluorescein-conjugated antibodies or similar reagents.
1.2.2.1.4.4

Tests in other cell cultures

Monolayers totalling at least 140 cm 2 are required. The cells must be frozen and thawed at
least 3 times and then centrifuged to remove cellular debris. Inoculate aliquots onto the
following cells at any time up to 70% confluency:

primary cells of the source species;

cells sensitive to viruses pathogenic for the species for which the vaccine is intended;

cells sensitive to pestiviruses.

The inoculated cells must be maintained in culture for at least 7 days, after which
freeze-thawed extracts should be prepared as above, and inoculated onto sufficient fresh
cultures of the same cell types of allow for the testing as described below. The cells are
incubated for at least a further 7 days.
All cultures must be regularly examined for the presence of any cytopathic changes
indicative of living organisms.
At the end of this period of 14 days, the inoculated cells must be subjected to the following
checks:

freedom from cytopathic and haemadsorbent organisms must be tested for using the
methods specified in paragraphs 1.2.2.1.4.1 and 1.2.2.1.4.2;

relevant substrates are tested for the absence of pestiviruses and other specific
contaminants by immunofluorescence as indicated in 1.2.2.1.4.3.

1.2.2.2

Identification of species

It must be shown that the MCS and the cells from the WCS at the highest passage level used
for production come from the species of origin specified by the manufacturer. This must be
demonstrated by one validated method.
When a fluorescence test is carried out and the corresponding serum to the species or origin
of cells is used and shows that all the tested cells are fluorescent, it is not necessary to carry
out other tests with reagents able to detect contamination by cells of other species.
1.2.2.3

Karyology

The cell lines used must be examined in the following manner:

A minimum of 50 cells undergoing mitosis must be examined in the MCS and a passage
level at least that of the highest to be used in production. Any chromosomal marker present
in the MCS must also be found in the high passage cells. The modal number of chromosomes
in these cells must not be more than 15% higher than that of the MCS. The karyotypes must be
identical. If the modal number exceeds the level stated, the chromosomal markers are not

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found in the WCS cells or the karyotype differs, the cell line may not be used for the
manufacture of biological products.

1.2.3 Requirements for primary cells

For most of the mammalian vaccines the use of primary cells is not acceptable for the
manufacture of vaccines. If a vaccine has to be produced on primary cells, they should be
obtained from a specific pathogen free herd or flock with complete protection from
introduction of diseases (e.g. disease barriers, filters on air inlets, no new animals
introduced without appropriate quarantine). In the case of chicken flocks these should comply
with the requirements of the European Pharmacopoeia monograph for SPF chickens. For a l l
other animals and species of birds, the herd or flock must be shown to be free from
appropriate pathogens. All the breeding stock in the herd or flock intended to be used to
produce primary cells for vaccine manufacture must be subject to a suitable regime such as
regular serological checks carried out at least twice a year and two supplementary
serological examinations performed in 15% of the breeding stock in the herd between the two
checks mentioned above.
Wherever possible, particularly for mammalian cells, a seed lot system should be used with,
for example, MCS formed from less than 5 passages, the WCS being no more than 5 passages
from the initial preparation of the cell suspension from the animal tissues.
Each MCS, WCS and cells of the highest passage of primary cells must be checked i n
accordance with Table 2 and the procedure described below. The sample tested will cover a l l
the sources of cells used for the manufacture of the batch. No batches of vaccine
manufactured using the cells may be marketed if any one of the checks performed produces
unsatisfactory results.
Table 2: Stages of primary cell culture at which testing shall be carried out

general microscopy
bacteria/fungi
mycoplasma
viruses
identification of species
1.2.3.1

MCS
+
+
+
+
+

WCS
+
+
+
+
-

Cells from WCS at highest passage level

+
-

Extraneous contaminants

1.2.3.1.1 General
The cells must be checked for their appearance under the microscope, for their rate of growth
and for other factors which will provide information on the state of health of the cells.
1.2.3.1.2 Bacteria and fungi
The cells must be checked for contamination with bacteria or fungi. Contaminated cells
must be discarded.
1.2.3.1.3 Mycoplasma
The cells must be checked for freedom from mycoplasma and pass the test for freedom from
mycoplasma.

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1.2.3.1.4 Viruses
The cells must not be contaminated by viruses and the checks must be performed in the
following manner:
The monolayers tested must be at least 70 cm 2 , prepared and maintained using a medium
and additives, and grown under similar conditions to those used for the preparation of the
biological product.
The monolayers must be maintained in culture for a total of at least 28 days. Subcultures
should be made at 7-day intervals, unless the cells do not survive for this length of time,
when the subcultures should be made on the latest day possible. Sufficient cells, in suitable
containers, must be produced for the final subculture to carry out the tests specified below.
The monolayers must be examined regularly throughout the incubation period for the
possible presence of cytopathic effects (cpe) and at the end of the observation period for cpe,
haemadsorbent viruses and specific viruses by immunofluorescence and other appropriate
tests as indicated below.
1.2.3.1.4.1

Detection of cytopathic viruses

Two monolayers of at least 6 cm2 each must be stained with an appropriate cytological stain.
Examine the entire area of each stained monolayer for any inclusion bodies, abnormal
numbers of giant cells or any other lesion indicative of a cellular abnormality which might
be attributable to a contaminant.
1.2.3.1.4.2

Detection of haemadsorbent viruses

Monolayers totalling at least 70 cm2 must be washed several times with an appropriate buffer
and a sufficient volume of a suspension of appropriate red blood cells added to cover the
surface of the monolayer evenly. After different incubation times examine cells for the
presence of haemadsorption.
1.2.3.1.4.3

Detection of specified viruses

Tests should be carried out for contaminants specific for freedom of the species of origin of
the cells and for the species for which the product is intended.
Sufficient cells on appropriate supports must be prepared to carry out tests for the agents
specified. Appropriate positive controls must be included in each test. The cells are subjected
to appropriate tests using fluorescein-conjugated antibodies or similar reagents.
1.2.3.1.4.4

Tests in other cell cultures

Monolayers totalling at least 140 cm 2 are required. The cells must be frozen then thawed at
least 3 times and then centrifuged to remove cellular debris. Inoculate aliquots onto the
following cells at any time up to 70% confluency:

cells sensitive to viruses pathogenic for the species for which the vaccine is intended;

cells sensitive to pestiviruses.

The inoculated cells must be maintained in culture for at least 7 days, after which
freeze-thawed extracts should be prepared as above and inoculated onto sufficient fresh
cultures of the same cell types to allow for the testing as described below. The cells are
incubated for at least a further 7 days.
All cultures must be regularly examined for the presence of any cytopathic changes
indicative of living organisms.

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At the end of this period of 14 days, the inoculated cells must be subjected to the following
checks:

freedom from cytopathic and haemadsorbent organisms must be tested for using the
methods specified in paragraphs 1.2.3.1.4.1 and 1.2.3.1.4.2;

relevant substrates are tested for the absence of pestiviruses and other specific
contaminants by immunofluorescence as indicated in 1.2.3.1.4.3.

1.2.3.2

Identification of species

It must be shown that the MCS comes from the species of origin specified by the manufacturer
(see Table 2). This must be demonstrated by one validated method.
When a fluorescence test is carried out and the corresponding serum to the species of origin
of cells is used and shows that all the tested cells are fluorescent, it is not necessary to carry
out other tests with reagents able to detect contamination by cells of other species.

1.2.4 Requirements for embryonated eggs

When vaccine organisms are grown in poultry embryos, such embryos may come from SPF
flocks or healthy non SPF flocks free from the presence of certain agents and their
antibodies as may be specified for a particular product. The inactivation process may have to
be shown to be effective against specified potential contaminants.
For the production of the MCS and for all passages up to the WCS, SPF eggs must be used.

1.2.5 Requirements for animals

Animals must be free from specific pathogens, as appropriate to the source species and the
target animal.

1.3

Virus seed

1.3.1 General requirements

Viruses used in manufacture shall be derived from a Seed Lot System. Each Master Seed
Virus (MSV) shall be tested as described below. A record of the origin, passage history
(including purification and characterisation procedures) and storage conditions shall be
maintained for each Seed Lot. Each MSV shall be assigned a specific code for identification
purposes. The MSV shall normally be stored in Aliquots at -70 C or lower if it is in liquid
form or at -20 C or lower if in a lyophilised form. Production of vaccine shall not normally
be undertaken using virus more than 5 passages from the MSV. In the tests described i n
section 1.3.3, 1.3.4 and 1.3.5, the organisms used shall not normally be more than 5 passages
from the MSV at the start of the tests unless otherwise indicated.
Where the MSV is contained within a permanently infected MCS, the following tests shall be
carried out on an appropriate volume of virus from disrupted MCS. Where relevant tests
have been carried out on disrupted cells to validate the suitability of the MCS, these tests need
not be repeated.

1.3.2 Propagation
The MSV and all subsequent passages shall be propagated on cells, on embryonated eggs or
in animals which have been shown to be suitable for vaccine production (see section 1.2) and
all such propagations shall only involve substance of animal origin that meet the
requirements of 1.1.

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1.3.3 Identity
The MSV shall be shown to contain only the virus stated. A suitable method shall be provided
to identify the vaccine strain and to distinguish it as far as possible from related strains.

1.3.4 Sterility and mycoplasma

The MSV shall pass the tests for sterility and freedom from mycoplasma.

1.3.5 Extraneous agents

Serum containing a high level of neutralising antibody to the virus of the Seed Lot shall be
prepared, using antigen that is not derived from any passage level of the virus isolate giving
rise to the MSV. Where it is not possible to prepare such a serum, other methods may be used
to remove selectively the virus of the seed lot.
Sera shall be prepared on a batch basis. Each batch shall be shown to be free of antibodies to
potential contaminants of the seed virus. Each batch shall be shown to be free of any
non-specific inhibition effects on the ability of viruses to infect and propagate within cells (or
eggs if applicable). Each batch shall be treated at 56 C for 30 minutes to inactivate
complement.
Using a minimum amount of serum prepared as above, a sample of the MSV shall be treated
so that all the vaccine virus is neutralised or removed. The final virus/serum mixture shall
contain at least the virus content of 10 dose of vaccine per ml if possible. The mixture should
then be tested for freedom from extraneous agents as follows:
The mixture shall be inoculated onto cultures of at least 70 cm2 of the required cell types. The
cultures may be inoculated at any stage of growth up to 70% confluency. At least one
monolayer of each type must be retained as a control. The cultures must be monitored daily
for a week. At the end of this period the cultures are freeze-thawed 3 times, centrifuged to
remove cell debris and reinoculated onto the same cell type as above. This is repeated twice.
The final passage must produce sufficient cells in appropriate vessels to carry out the tests
below.
Cytopathic and haemadsorbing agents are tested for using the methods described i n
paragraphs 1.2.2.1.4.1 and 1.2.2.1.4.2. Techniques such as immunofluorescence should be used
for detection of specific contaminants as described in paragraphs 1.2.2.1.4.3. The MSV i s
inoculated onto:

primary cells of the species or origin of the virus;

cells sensitive to viruses pathogenic for the species for which the vaccine is intended.

cells sensitive to pest viruses.

If the MSV is shown to contain living organisms of any kind, other than virus of the species
and strain stated, then it is unsuitable for vaccine production.

1.4 Bacterial seed

1.4.1 General requirements
The bacteria used in the vaccine shall be stated by genus and species (and varieties where
appropriate). The origin, date of isolation and designation of the bacterial strains used shall
be given, and details provided, where possible, of the passage history, including details of the
media used at each stage. Bacteria used in manufacture shall be derived from a Seed Lot
System wherever possible. Each Master Seed Lot, (henceforth known as Seed Lot) shall be

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tested as described below. A record of the origin, passage history (including purification and
characterisation procedures) and storage conditions shall be maintained for each Seed Lot.
Each Seed Lot shall be assigned a specific code for identification purposes.

1.4.2 Identity and purity

Each Seed Lot shall be shown to contain only the species and strain of bacterium stated. A
description of the method of identifying each strain by biochemical, serological and
morphological characterisations and distinguishing it as far as possible from related
strains shall be provided, as shall also the methods of determining the purity of the strain. If
the Seed Lot is shown to contain living organisms of any kind other than the species and
strain stated, then it is unsuitable for vaccine production.

1.4.3 Seed lot requirements

The minimum and maximum number of subcultures of each Seed Lot prior to the production
stage shall be specified. The methods used for the preparation of seed cultures, preparation of
suspension for seeding, techniques for inoculation of seeds, titre and concentration of
inocula and the media used shall be described. It shall be demonstrated that the
characteristics of the seed material (e.g. dissociation or antigenicity) are not changed by
these subcultures.
The conditions under which each seed lot is stored shall be described.

1.5 Media for bacterial vaccines

At least the qualitative composition should be given of media used for seed culture
preparation and for production. Named ingredients should be specified as to grade. Where
ingredients are claimed as proprietary, this should be indicated and an appropriate
description given. Ingredients which are derived from animals should be specified as to the
species source and country of origin, and must comply with the criteria described in section
1.1. Preparation processes for media used, including sterilisation procedures shall be
described.

1.6

Antibiotics

The addition of antibiotics in the process of the manufacture of the product shall normally be
restricted to cell culture fluids and other media, egg inocula and material harvested from
skin or other tissues.
Not more than three antibiotics shall be permitted for simultaneous use for these purposes.
Penicillin and Streptomycin are not allowed for vaccines used by parenteral or aerosol
application. If the antibiotics used are not recommended for use in the target species, they
shall be shown to have no harmful effect on the vaccinated animals.
Antibiotics shall not be added to the finished product.

1.7

Preservatives

The efficacy of preservatives in multidose containers should be demonstrated. The

concentration of the preservative in the final filled vaccine and its persistence throughout
shelf life must be checked.
If no preservative is included, the applicant should demonstrate that the product remains
acceptable for its recommended period of use after broaching the vial.

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1.8 Other substances

All other substances used in vaccine production shall be prepared in such a way as to prevent
contamination of the vaccine with any living organism or toxin.

1.9 Manufacturing process Inactivation

The testing of the inactivation kinetics described below is required to be done once. The rest
of this section applies each production run.

1.9.1 Inactivated virus vaccines

1.9.1.1

Inactivation kinetics

The inactivating agent and the inactivation procedure shall be shown, under conditions of
vaccine manufacture, to inactivate the vaccine virus. Adequate data on inactivation kinetics
shall be provided. Normally the virus shall be shown to be inactivated within a timeperiod
equivalent to not more than 67% of the inactivation process used during manufacture.
1.9.1.2

Prerequisite to inactivation

Prior to inactivation, care should be taken to ensure a homogeneous suspension, free from
particles that may not be penetrated by the inactivating agent.
1.9.1.3

Aziridine

If an aziridine compound is used as the inactivating agent then it shall be shown that no
inactivating agent remains at the end of the inactivation procedure. This may be
accomplished by neutralising the inactivating agent with thiosulphate and demonstrating
residual thiosulphate in the bulk harvest at the completion of the inactivation procedure.
1.9.1.4

Formaldehyde

If formaldehyde is used as the inactivating agent then a test for free formaldehyde should be
carried out. Not more than 0.05% of free formaldehyde shall be present in the vaccine unless
this higher concentration has been shown to be safe.
1.9.1.5

Betapropriolactone

Where betapropriolactone (BPL) is used as the inactivant, it shall be shown, at the end of the
inactivation procedure, that there remains no significant amount of BPL in the inactivated
bulk.
1.9.1.6

Other methods

When other inactivation methods are used, appropriate tests should be carried out to
demonstrate that the inactivant has been removed or depleted or any residues are safe.
1.9.1.7

Inactivation testing

A test for complete inactivation shall be performed on the harvest immediately after the
inactivation procedure and, if applicable, the neutralisation or removal of the inactivating
agent. The test selected should be appropriate to the vaccine virus being used and should
consist of at least two passages in cells, embryonated eggs or where necessary in animals.
The number of cell samples, eggs or animals should be sufficient to ensure appropriate
sensitivity of the test. For cell cultures, at least 150 cm 2 of cell culture monolayer shall be
inoculated with 1.0 ml of harvest. No evidence of the presence of any live virus or
microorganism should be observed.

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1.9.2 Bacterial inactivated vaccines

1.9.2.1

Inactivation kinetics

The test in 1.9.1.1 shall be performed. Normally the period of inactivation used in production
shall exceed the time shown to be adequate by the inactivation kinetics by at least 33%.
1.9.2.2

Prerequisite to inactivation

Prior to inactivation, care should be taken to ensure a homogeneous suspension, free from
particles that may not be penetrated by the inactivating agent.
1.9.2.3

Aziridine

If an aziridine compound is used as the inactivating agent then it shall be shown that no
inactivating agent remains at the end of the inactivation procedure. This may be
accomplished by neutralising the inactivating agent with thiosulphate and demonstrating
residual thiosulphate in the bulk harvest at the completion of the inactivation procedure.
1.9.2.4

Formaldehyde

If formaldehyde is used as the inactivating agent then a test for free formaldehyde should be
carried out. Not more than 0.05% of free formaldehyde shall be present in the vaccine unless
this higher concentration has been shown to be safe.
1.9.2.5

Inactivation testing

A test for complete inactivation shall be performed on the harvest immediately after the
inactivation procedure and, if applicable, the neutralisation or removal of the inactivating
agent. The test selected should be appropriate to the vaccine bacteria used and should consist
of at least two passages in production media or in media prescribed in the European
Pharmacopoeia.
No evidence of any live microorganism should be observed.

1.10 Samples
Samples of all seed materials, reagents, in-process material and finished product shall be
supplied to competent authorities, on request.

2.

FINISHED PRODUCT ASSAY RESULTS REQUIRED IN

THE APPLICATION FOR MARKETING
AUTHORISATION

For each application, the results of the following tests shall be presented.

2.1 Safety
Safety testing shall be carried out as specified in Directive 81/852/EEC, as amended by
Directive 92/18/EEC and the indications given below.
The dose to be used shall be that quantity of the product to be recommended for use and
containing the maximum titre or potency for which the application is submitted.
The samples for the safety testing shall be taken from a batch or batches produced according
to the manufacturing process described in the application for marketing authorisation.

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2.1.1 Safety of the administration of one dose

In test C 12, the rectal temperatures should be recorded on at least the day before vaccination
and the following four days, in a sufficient number of animals.

2.1.2 Safety of one administration of an overdose

In test C 2 a double dose should normally be administered.

2.1.3 Field trials

During any field trial undertaken in accordance with part D of the Annex of Directive
81/852/EEC, as amended by Directive 92/18/EEC, the studies should include measurement of
the rectal temperatures of a sufficient number of animals, before and after vaccination. The
size and persistence of any local reaction and the proportion of animals showing local or
systemic reactions shall be recorded. Performance measurements should be made, where
appropriate.

2.2 Efficacy
Efficacy testing shall be carried out as specified in Directive 81/852/EEC, as amended by
Directive 92/18/EEC and the indications given below.
The dose to be used shall be that quantity of the product to be recommended for use and
containing the minimum titre or potency for which the application is submitted.
The samples for the efficacy testing shall be taken from a batch or batches produced
according to the manufacturing process described in the application for marketing
authorisation.
The efficacy evidence must support all the claims being made. For example, claims for
protection against respiratory disease must be supported by at least evidence of protection
from clinical signs of respiratory disease. Where it is claimed that there is protection from
infection this must be demonstrated using reisolation techniques. If more than one claim i s
being made, supporting evidence for each will be required.
Real time unvaccinated controls will be required.
Studies of immunological compatibility shall be undertaken when simultaneous
administration is recommended either by the applicant or in a usual vaccination schedule.

2.3 Stability
Evidence of stability shall be presented to justify the shelf life. The evidence shall take the
form of the results of:
a)

potency tests (see 3.5) carried out at intervals until 3 months beyond the requested shelf
life on at least 3 batches of vaccine prepared from three consecutive production runs
kept under recommended storage conditions. These three production runs may be
carried out on a pilot scale, providing this mimics the full-scale production described
in the application,
and

b)

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tests described under points 3.6 to 3.9.

All numbering in sections 2.1.1 to 2.1.3 refers to the corresponding sections in the Annex of Directive
81/852/EEC, as amended by Directive 92/18/EEC.

____________________________________________________________ 7BIm2a

A short shelf life will be granted, if necessary, while this evidence is collected.
Where a finished product requires reconstitution prior to administration, the vaccine shall
be reconstituted with the diluent as recommended and the resulting mixture titrated or tested
for potency immediately after reconstitution and again after storage.

2.4 Batch quality control results

Three sets of results of the quality control test, outlined below, must be presented in the
dossier. The results must be obtained from tests on three batches from three consecutive
production runs produced according to the production process for the product, described in the
application for marketing authorisation.

3.

FINISHED PRODUCT BATCH TESTING

The tests in this section shall normally be performed on each batch or subbatch of vaccine
produced. In the case of subbatches which differ only due to their processing after bulk
blending, for example in their filling session or vial size, some tests may be carried out on
the final bulk or on one of the subbatches.
The applicant must have demonstrated that the subsequent procedure does not result i n
differences in test results and the results obtained from tests on the bulk can be reproduced
on the subbatch(es) of the finished product. For example, it may be expected that tests of
potency of liquid inactivated vaccines can be done on the bulk. On the other hand, tests for
sterility must be carried out on each subbatch.

3.1 Identification
Tests for identification shall be carried out where this information cannot be obtained from
other tests, e.g. ELISA.

3.2 Safety and extraneous antigens

The vaccine shall be shown to be safe in two susceptible animals of one species and one class
for which the vaccine is recommended. The animals are given a double dose of vaccine.
Rectal temperatures and any adverse local or systemic reactions should be recorded.
Where required in specific Ph. Eur. monographs:
a)

the double dose shall be followed by administration of a single dose 14 days later;
and/or

b)

serum samples shall be obtained from the animals two weeks after the last vaccination
and tested for the absence of antibodies to organisms pathogenic for the species and
antibodies to other organisms handled on the premises. Additional tests may be
imposed at times of serious disease in the vicinity of the manufacturing premises. It i s
the responsibility of the manufacturer to notify the competent authorities of the
circumstances.

3.3 Sterility
The vaccine shall be shown to be sterile.

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3.4 Inactivation
A suitable test for complete inactivation of vaccine organisms shall be carried out on the
finished vaccine. The protocol for this test shall normally be the same as that for the tests
performed on the harvest (see 1.9.1.7). If the presence of adjuvant or other substances render
this impossible then the test shall be performed on a sample of bulk organism harvest taken
immediately before the addition of the adjuvant. Bulk antigen so sampled shall not be stored
except in the vessel from which the sample was taken.

3.5 Potency
The vaccine shall be shown to be of satisfactory potency using validated methods.

3.6 Physical Tests

Oil adjuvanted vaccine shall be tested for viscosity by a suitable method. The stability of the
emulsion shall be demonstrated.

3.7 Chemical Tests

Tests for the concentrations of appropriate substances such as aluminium and preservatives
shall be carried out to show that these are in conformity with the limits set for the product.

3.8 Moisture content

The moisture content of freeze-dried products shall be determined and shown to be within the
limits set for the product.

3.9 pH
The pH of liquid products shall be measured and shown to be within the limits set for the
product.

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