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Comparison of requirements in the European Union and United States of America for pre-clinical viral safety testing of veterinary vaccines
Biologicals 38 (2010) 340e345
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Comparison of requirements in the European Union and United States of America for pre-clinical viral safety testing of veterinary vaccines Sarah Sheridan*, Julie Coughlin BioReliance Ltd., Todd Campus, West of Scotland Science Park, Glasgow G20 0XA, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2010 Accepted 10 January 2010
Of paramount importance in ensuring the safety of live and inactivated veterinary vaccines is demonstration of freedom from extraneous agents in biological starting materials used in their production. Both the European Union (EU) and United States of America (US) provide regulations and guidelines on extraneous agent testing of veterinary vaccines including guidance from the Committee for Medicinal Products for Veterinary Use (CVMP), the European Pharmacopoeia (Ph. Eur.) and the USDA Code of Federal Regulations, Title 9 (9CFR). There are distinct requirements prescribed in EU and US regulations and guidelines. The differences in EU and US requirements for extraneous agent testing of starting materials are such that there may be occasions when no one test may satisfy both sets of regulations for a given scenario. For compliance with both, for global licensing purposes it may therefore be necessary to perform additional tests and/or to justify methods chosen from one set of regulations over another, based on a variety of factors. Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
Keywords: Veterinary vaccines Extraneous agents European Pharmacopeia CVMP USDA 9CFR
1. Introduction As part of safety testing requirements, biological substrates and ingredients and products of animal origin used in the production of veterinary vaccines must be shown to be free from contaminants including viruses, bacteria, fungi and mycoplasma. In Europe, regulations on the safety testing of veterinary vaccines are laid out in the European Pharmacopeia (Ph. Eur.) [1]. Guidance on the regulations governing veterinary medicinal products in the European Union (EU) can be found in various guidelines prepared by the Committee for Medicinal Products for Veterinary Use (CVMP) [2e5]. Together, these documents provide a generally harmonised approach to safety testing requirements within the EU. In the United States of America (US) veterinary vaccines are regulated by the Center for Veterinary Biologics (CVB), Animal and Plant Health Inspection Service (APHIS), US Department of Agriculture (USDA) where regulations for safety testing of raw materials used in the production of veterinary vaccines are laid out in the Code of Federal Regulations, Title 9; Animals and Animal Products (9 CFR) [6]. These regulations are distinct from the EU requirements and include some different methodologies and recommendations for safety testing.
* Corresponding author. Tel.: þ44 141 946 9999; fax: þ44 141 946 0000. E-mail address: [email protected] (S. Sheridan).
In the animal health industry it is not unusual that a product license is desired for both the European and US market. Accordingly, a comprehensive testing strategy may be necessary if the safety issues relating to species of origin and target species for starting materials are to be addressed for both EU and US regulatory review. Typically the scope of pre-clinical extraneous agent testing will include cell and virus seeds and substances of animal origin. The relative differences between EU and US requirements for the stages at which these starting materials should be tested for all extraneous agents are shown in Table 1. 2. Starting materials For the detection of viral extraneous agents in starting materials, suitably prepared test materials are propagated in culture to allow amplification of viral contaminants on a range of detector cell lines. Depending on the test material, these cell lines will include some or all of the following: primary cells and/or cells of the source species (Ph. Eur., CVMP, 9CFR); cells sensitive to viruses pathogenic for the species for which the vaccine is intended (Ph. Eur., CVMP, 9CFR); cells sensitive to Pestiviruses (Ph. Eur., CVMP); cells of the species of the cell line in which the vaccine is produced (9CFR); cells of bovine origin (9CFR) and vero cells (9CFR). A detector cell line is chosen, for each of the above categories, based on its sensitivity to the viruses of concern. Reference to the CVMP tables of extraneous agents provides guidance on what
1045-1056/$36.00 Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2010.01.001
S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345 Table 1 Stages at which starting materials should be tested; comparison of requirements in the EU and the US. Testing required
Master cell seed
Working cell seed (WCS)
WCS at highest passage level
Master virus seed
Substances of animal origin
Bacteria/fungi Mycoplasma Viruses
þþa þþ þþ
þb þ þ
þc þ d
þþ þþ þþ
þþ þþ þþ
a b c d
EU and US requirement. EU requirement. US requirement. Not required.
agents should be tested for depending on the species concerned and whether general methods, such as staining for cytopathic effect, or specific methods should be used [7]. In the 9CFR, lists of viruses that should be tested for by specific means only are included for a similar range of species, with the exception of rodents and lagomorphs. Cultures are maintained for a minimum specified period with regular subculture and observation for cytopathic effects (CPE) and morphological change. During and/or following the culture period, endpoint tests for viral contaminants are performed on the detector cells. These include general methods such as cytological staining and haemadsorption assay (HA) in addition to specific tests such as immunofluorescence assay (IFA). Where in vitro tests are not considered to be sensitive enough for the virus of concern, alternative detection methods may be used such as nucleic acid tests, in vivo tests and tests in eggs. A comparison of the differences between EU and US regulatory methods for extraneous agent testing of cell seeds, virus seeds and substances of animal origin follows in Sections 2.1e2.3.
341
extraneous agents. Tests for freedom from contaminating viruses are carried out directly on cultures of MCS and WCS and also by inoculating MCS and WCS extracts onto suitable detector cell lines as described below. Monolayers of MCS and WCS for testing should be at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) and cultured directly using conditions similar to those used for preparation of the vaccine for at least 21 days (9CFR) or 28 days (Ph. Eur., CVMP) (Fig. 1). Subcultures are done typically every 7 days throughout the cultivation period with regular observations for evidence of cytopathic agents. At the end of the cultivation period monolayers of specified surface area are tested for cytopathic agents by staining, for haemadsorbing agents by HA and for specified viruses by IFA or similar specific tests. A comparison of the differences between EU and US regulatory methods for these endpoint tests is detailed in Section 3. At the conclusion of the MCS and WCS direct test, monolayers of at least 140 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of these cells are freeze/thawed at least three times and clarified by centrifugation. Aliquots of the resulting extract are then inoculated onto monolayers of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of suitable detector cells. The inoculated cells are cultured for at least 14 days with at least one subculture (9CFR) or by preparation of freeze/thawed extracts with inoculation onto fresh monolayers of corresponding cell type after at least 7 days (Ph. Eur., CVMP) (Fig. 2). The inoculated cultures are observed regularly for evidence of cytopathic agents. After at least 14 days post inoculation (d.p.i.) monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and for specified viruses, by IFA or similar specific tests (see section 3.0 for details on EU and US differences). 2.2. Virus seeds
2.1. Cell banks Master cell seeds (MCS) and working cell seeds (WCS) used in the production of veterinary vaccines must be tested to preclude
Master seed viruses for mammalian veterinary vaccines must also be tested to preclude extraneous agents. The virus may need to be neutralised so that the virus seed can be tested for extraneous
Fig. 1. Comparison of EU and US methods for extraneous agent testing of cell banks: direct test. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
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S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345
Fig. 2. Comparison of EU and US methods for extraneous agent testing of cell banks: extract test. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
viruses in the appropriate cell culture system. Neutralisation is performed to prevent infection and propagation of the virus seed in the detector cells. Polyclonal or monoclonal antibody preparations containing neutralising antibodies to the virus seed must be prepared using an antigen distinct from the virus seed isolate. The neutralising antiserum must be shown to be free from antibodies to potential contaminants of the virus seed and also from any nonspecific inhibitory effects on the ability of contaminating viruses to infect and propagate within the cell culture. The antiserum should be used in a minimal volume to neutralise, if possible, at least the virus content of 10 doses of vaccine per ml (Ph. Eur., CVMP). Alternative methods to neutralise or remove the test virus may be used in the absence of a suitable neutralising antiserum. Virus (neutralised, if required) is inoculated onto monolayers of suitable detector cells of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) (Fig. 3). The inoculated cells are cultured for at least 28 days (Ph. Eur., CVMP) or 14 days (9CFR). The cells are subcultured by preparation of freeze/thawed extracts with inoculation onto fresh monolayers of corresponding cell type (Ph. Eur., CVMP) or by at least one subculture (9CFR). The inoculated cultures are observed regularly for evidence of cytopathic agents. After at least 28 d.p.i. (Ph. Eur., CVMP) or 14 d.p.i. (9CFR), monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests (see Section 3 for details on EU and US differences). 2.3. Substances of animal origin Substances of animal origin may be used during the manufacture of veterinary vaccines such as serum and trypsin used in the
cultivation of cell seeds. These raw materials must be tested for freedom from extraneous agents once they have been prepared in an appropriate manner for inoculation onto detector cell cultures. For the detection of extraneous viruses, test substances must be suitably prepared. Solids are dissolved or resuspended in a suitable medium to create a solution or suspension containing at least 300 g/L of the test material (Ph. Eur., CVMP) or 3.75 ml or 15% of the ingredient in growth medium (9CFR). The test material is inoculated onto monolayers of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of suitable detector cells. The inoculated cells are cultured for a total of at least 21 days (Ph. Eur., CVMP and 9CFR) with regular observations for cytopathic agents (Fig. 4). At the end of each 7 day period, monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests. The remaining cells are subcultured (Ph. Eur., CVMP). Alternatively, cells are subcultured at least two times over the 21 day period, at the end of which monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests (9CFR). In the EU, requirements for testing bovine serum used in the production of veterinary vaccines is outlined in CVMP guidance on “Requirements and controls applied to bovine serum used in the production of immunological veterinary medicinal products” [8]. General and specific tests should be performed prior to any serum inactivation treatment and further tests performed postinactivation. The general and specific tests applied must be capable of detecting specific viruses listed within the guidance document.
S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345
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Fig. 3. Comparison of EU and US methods for extraneous agent testing of virus seeds. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
3. Endpoint tests 3.1. Staining for cytopathic effects Cells infected with cytopathic viruses display CPE or morphological changes. Cells exhibiting these changes can be fixed and stained with an appropriate cytological stain such as Giemsa and hematoxylin and eosin (H&E). In accordance with EU and US regulations, at least two monolayers of 6 cm2 (Ph. Eur., CVMP) or at least one monolayer of 6 cm2 (9CFR) of cells are stained. Cells are observed for inclusion bodies, giant cells or other abnormalities or lesions indicative of changes in cellular morphology which may be attributable to a viral contaminant. 3.2. Haemadsorption assay Some members of the virus groups of Orthomyxo-, Toga-, Parvoand Paramyxo-viruses may not always produce an obvious CPE in susceptible cell cultures. These viruses may be detected by a haemadsorption assay that utilises their haemagglutinin expression. Monolayers of at least 70 cm2 (Ph. Eur., CVMP) or at least one monolayer of 6 cm2 (9CFR) of cells are required. A suspension (0.2% as per 9CFR) of red blood cells including chicken, guinea pig and any species of concern is added to the test monolayer (human red blood cells may also be included). Cultures are incubated at 4 C, room temperature and 37 C (Ph. Eur., CVMP) or 4 C and
20e25 C (9CFR) for a period of 30 min and/or 60 min. The cultures are then washed and observed microscopically for evidence of haemadsorption. 3.3. Immunofluorescence assay Monoclonal or polyclonal antibodies specific for particular antigens are utilised to detect specific viruses in fixed cell cultures. The antibodies are labelled with a fluorescent dye and, when they are in contact with their specific antigen, the reaction site can be observed using a fluorescent microscope. If viral antigen is not present the antibodies are removed during the washing steps. This test is suitable for detecting non-cytopathic and non-haemadsorbing viruses. The specific viruses to be tested for by IFA are determined by the species of origin of the cell seed or virus seed under test and the intended species of the product. Monolayers of sufficient cells (Ph. Eur., CVMP) or at least 6 cm2 of cells under test (9CFR) are required. A positive control inoculated with 100e300 FAID50 (50% fluorescent antigen infectious doses) should be included and may be fixed for IFA prior to day 7 of the culture period if fluorescence is enhanced by doing so, provided that a test monolayer is also fixed at the same time, as well as at least 7 days after subculturing (9CFR). Regardless of the species of origin and target species, monolayers should be tested for Pestiviruses (Ph. Eur., CVMP) or bovine viral diarrhoea virus, reovirus and rabies virus (9CFR).
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Fig. 4. Comparison of EU and US methods for extraneous agent testing of substances of animal origin. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
4. Avian vaccines Virus and cell seeds used in the production of veterinary vaccines for avian species must be tested to preclude extraneous agents. For the detection of avian derived contaminants, in vivo and egg tests are recommended in addition to in vitro assays using suitably sensitive avian detector cell lines. A full range of assays to detect avian viral agents would include the following tests: extraneous viruses using fertilized eggs (Ph. Eur., CVMP, 9CFR); extraneous viruses using cell cultures (Ph. Eur., CVMP); avian leucosis viruses (Ph. Eur., CVMP, 9CFR); avian reticuloendotheliosis virus (Ph. Eur., CVMP, Veterinary Services (VS) Memorandum 800.88); extraneous agents using chicks (Ph. Eur., CVMP, 9CFR); and chick anaemia virus (Ph. Eur., CVMP, VS Memorandum 800.89). Endpoint tests are based on observations for abnormalities and mortality (in vivo or egg tests) or CPE and HA (in vitro tests). Specific tests such as IFA, enzyme-linked immunosorbent assay (ELISA) or the complement fixation test for avian leucosis (COFAL) for viruses and other pathogens associated with avian species (e.g. chicken, duck, turkey or goose) are typically performed as endpoint tests for either the in vitro or in vivo assays [4].
As for mammalian virus seeds, neutralisation of the virus may be required for testing avian virus seeds for extraneous viruses in the various test systems prescribed. Polyclonal or monoclonal antibody is produced, as outlined for mammalian vaccines. The neutralising antiserum must be shown to be free from antibodies against, and free from inhibitory effects on, agents listed within the relevant regulations and guidelines [1,4]. Monospecific antisera for virus neutralisation are not required to be tested for antibodies against all of these viruses, if it can be shown that the immunizing antigen could not have been contaminated with antigens derived from the virus in question and if the virus is not known to infect the species of origin of the serum. Also it is not necessary to retest sera obtained from birds from specific pathogen free (SPF) chicken flocks (Ph. Eur). Batches of serum must not be prepared from any passage level derived from the virus isolate used to prepare the master seed lot or from an isolate cultured in the same cell line. The antiserum should be used minimally to neutralise (if possible) at least the virus content of 10 doses of vaccine per 0.1 ml (in vitro tests) or 0.2 ml (in vivo tests). Alternative methods for neutralising or removing the test virus may be used in the absence of a suitable neutralising antiserum.
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5. Fish vaccines
Acknowledgments
Virus and cell seeds used in the production of veterinary vaccines intended for fish must also be tested to preclude extraneous agents. Guidance provided by the CVMP recommends that testing be conducted as described for starting materials of mammalian origin. Endpoint tests using specific methods such as IFA should be included for the detection of pathogens of piscine origin [5].
We thank Doris Barnett, BioReliance Corporation, Rockville, Maryland, USA and Donna Gatewood, D.V.M., M.S, Center for Veterinary Biologics, Ames, Iowa, USA.
6. Discussion There are differences between EU and US requirements for extraneous agent testing of starting materials used in the production of veterinary vaccines. There are differences with respect to what stage starting materials should be tested at, as well as the specific methods that should be employed for these tests. In addressing concerns about extraneous agents for specific species, further differences exist; under EU requirements, lists of viruses that should be tested for by both general and specific methods are included whereas under US requirements, only viruses for which specific detection methods are required are listed. Furthermore, the species for which such lists are included are not the same; EU requirements include lists of extraneous agents of rodent, lagomorph and fish origin that are not included in US requirements (although there could be a requirement under US regulations to test for extraneous agents of these species if involved). Where lists for the same species do exist in EU and US regulations and guidelines, the viruses contained in these lists are not identical.
References [1] European Pharmacopoeia. In: Methods of analysis. 6th ed. Strasbourg: Council of Europe; 2009. [2] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality. vol. 7B, General requirements for the production and control of live mammalian bacterial and viral vaccines for veterinary use; March 1992 [7BIm1a (III/3182/91-EN)]. [3] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality. vol. 7B, General requirements for the production and control of inactivated mammalian bacterial and viral vaccines for veterinary use. ; March 1992 [7BIm1b (III/3181/ 91-EN)]. [4] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality, vol. 7B. Specific requirements for the production and control of avian live and inactivated viral and bacterial vaccines; November 1992 [7BIm3a (III/3363/92-EN)]. [5] Specific requirements for the production and control of live and inactivated vaccines intended for fish; September 1994 [7BIm9a (III/3590/92-EN)]. [6] Code of federal regulations title 9. Animals and animal products, Part 113 standard requirements; January 2009. [7] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality, vol. 7B, Table of extraneous agents to be tested for in relation to the general and species specific guidelines on production and control of mammalian veterinary vaccines; September 1994 [7BIm10a]. [8] Revised guideline on requirements and controls applied to bovine serum used in the production of immunological veterinary medicinal products. EMEA/ CVMP/743/00-Rev. 2; November 2005.
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Comparison of requirements in the European Union and United States of America for pre-clinical viral safety testing of veterinary vaccines Sarah Sheridan*, Julie Coughlin BioReliance Ltd., Todd Campus, West of Scotland Science Park, Glasgow G20 0XA, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2010 Accepted 10 January 2010
Of paramount importance in ensuring the safety of live and inactivated veterinary vaccines is demonstration of freedom from extraneous agents in biological starting materials used in their production. Both the European Union (EU) and United States of America (US) provide regulations and guidelines on extraneous agent testing of veterinary vaccines including guidance from the Committee for Medicinal Products for Veterinary Use (CVMP), the European Pharmacopoeia (Ph. Eur.) and the USDA Code of Federal Regulations, Title 9 (9CFR). There are distinct requirements prescribed in EU and US regulations and guidelines. The differences in EU and US requirements for extraneous agent testing of starting materials are such that there may be occasions when no one test may satisfy both sets of regulations for a given scenario. For compliance with both, for global licensing purposes it may therefore be necessary to perform additional tests and/or to justify methods chosen from one set of regulations over another, based on a variety of factors. Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
Keywords: Veterinary vaccines Extraneous agents European Pharmacopeia CVMP USDA 9CFR
1. Introduction As part of safety testing requirements, biological substrates and ingredients and products of animal origin used in the production of veterinary vaccines must be shown to be free from contaminants including viruses, bacteria, fungi and mycoplasma. In Europe, regulations on the safety testing of veterinary vaccines are laid out in the European Pharmacopeia (Ph. Eur.) [1]. Guidance on the regulations governing veterinary medicinal products in the European Union (EU) can be found in various guidelines prepared by the Committee for Medicinal Products for Veterinary Use (CVMP) [2e5]. Together, these documents provide a generally harmonised approach to safety testing requirements within the EU. In the United States of America (US) veterinary vaccines are regulated by the Center for Veterinary Biologics (CVB), Animal and Plant Health Inspection Service (APHIS), US Department of Agriculture (USDA) where regulations for safety testing of raw materials used in the production of veterinary vaccines are laid out in the Code of Federal Regulations, Title 9; Animals and Animal Products (9 CFR) [6]. These regulations are distinct from the EU requirements and include some different methodologies and recommendations for safety testing.
* Corresponding author. Tel.: þ44 141 946 9999; fax: þ44 141 946 0000. E-mail address: [email protected] (S. Sheridan).
In the animal health industry it is not unusual that a product license is desired for both the European and US market. Accordingly, a comprehensive testing strategy may be necessary if the safety issues relating to species of origin and target species for starting materials are to be addressed for both EU and US regulatory review. Typically the scope of pre-clinical extraneous agent testing will include cell and virus seeds and substances of animal origin. The relative differences between EU and US requirements for the stages at which these starting materials should be tested for all extraneous agents are shown in Table 1. 2. Starting materials For the detection of viral extraneous agents in starting materials, suitably prepared test materials are propagated in culture to allow amplification of viral contaminants on a range of detector cell lines. Depending on the test material, these cell lines will include some or all of the following: primary cells and/or cells of the source species (Ph. Eur., CVMP, 9CFR); cells sensitive to viruses pathogenic for the species for which the vaccine is intended (Ph. Eur., CVMP, 9CFR); cells sensitive to Pestiviruses (Ph. Eur., CVMP); cells of the species of the cell line in which the vaccine is produced (9CFR); cells of bovine origin (9CFR) and vero cells (9CFR). A detector cell line is chosen, for each of the above categories, based on its sensitivity to the viruses of concern. Reference to the CVMP tables of extraneous agents provides guidance on what
1045-1056/$36.00 Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2010.01.001
S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345 Table 1 Stages at which starting materials should be tested; comparison of requirements in the EU and the US. Testing required
Master cell seed
Working cell seed (WCS)
WCS at highest passage level
Master virus seed
Substances of animal origin
Bacteria/fungi Mycoplasma Viruses
þþa þþ þþ
þb þ þ
þc þ d
þþ þþ þþ
þþ þþ þþ
a b c d
EU and US requirement. EU requirement. US requirement. Not required.
agents should be tested for depending on the species concerned and whether general methods, such as staining for cytopathic effect, or specific methods should be used [7]. In the 9CFR, lists of viruses that should be tested for by specific means only are included for a similar range of species, with the exception of rodents and lagomorphs. Cultures are maintained for a minimum specified period with regular subculture and observation for cytopathic effects (CPE) and morphological change. During and/or following the culture period, endpoint tests for viral contaminants are performed on the detector cells. These include general methods such as cytological staining and haemadsorption assay (HA) in addition to specific tests such as immunofluorescence assay (IFA). Where in vitro tests are not considered to be sensitive enough for the virus of concern, alternative detection methods may be used such as nucleic acid tests, in vivo tests and tests in eggs. A comparison of the differences between EU and US regulatory methods for extraneous agent testing of cell seeds, virus seeds and substances of animal origin follows in Sections 2.1e2.3.
341
extraneous agents. Tests for freedom from contaminating viruses are carried out directly on cultures of MCS and WCS and also by inoculating MCS and WCS extracts onto suitable detector cell lines as described below. Monolayers of MCS and WCS for testing should be at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) and cultured directly using conditions similar to those used for preparation of the vaccine for at least 21 days (9CFR) or 28 days (Ph. Eur., CVMP) (Fig. 1). Subcultures are done typically every 7 days throughout the cultivation period with regular observations for evidence of cytopathic agents. At the end of the cultivation period monolayers of specified surface area are tested for cytopathic agents by staining, for haemadsorbing agents by HA and for specified viruses by IFA or similar specific tests. A comparison of the differences between EU and US regulatory methods for these endpoint tests is detailed in Section 3. At the conclusion of the MCS and WCS direct test, monolayers of at least 140 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of these cells are freeze/thawed at least three times and clarified by centrifugation. Aliquots of the resulting extract are then inoculated onto monolayers of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of suitable detector cells. The inoculated cells are cultured for at least 14 days with at least one subculture (9CFR) or by preparation of freeze/thawed extracts with inoculation onto fresh monolayers of corresponding cell type after at least 7 days (Ph. Eur., CVMP) (Fig. 2). The inoculated cultures are observed regularly for evidence of cytopathic agents. After at least 14 days post inoculation (d.p.i.) monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and for specified viruses, by IFA or similar specific tests (see section 3.0 for details on EU and US differences). 2.2. Virus seeds
2.1. Cell banks Master cell seeds (MCS) and working cell seeds (WCS) used in the production of veterinary vaccines must be tested to preclude
Master seed viruses for mammalian veterinary vaccines must also be tested to preclude extraneous agents. The virus may need to be neutralised so that the virus seed can be tested for extraneous
Fig. 1. Comparison of EU and US methods for extraneous agent testing of cell banks: direct test. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
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S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345
Fig. 2. Comparison of EU and US methods for extraneous agent testing of cell banks: extract test. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
viruses in the appropriate cell culture system. Neutralisation is performed to prevent infection and propagation of the virus seed in the detector cells. Polyclonal or monoclonal antibody preparations containing neutralising antibodies to the virus seed must be prepared using an antigen distinct from the virus seed isolate. The neutralising antiserum must be shown to be free from antibodies to potential contaminants of the virus seed and also from any nonspecific inhibitory effects on the ability of contaminating viruses to infect and propagate within the cell culture. The antiserum should be used in a minimal volume to neutralise, if possible, at least the virus content of 10 doses of vaccine per ml (Ph. Eur., CVMP). Alternative methods to neutralise or remove the test virus may be used in the absence of a suitable neutralising antiserum. Virus (neutralised, if required) is inoculated onto monolayers of suitable detector cells of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) (Fig. 3). The inoculated cells are cultured for at least 28 days (Ph. Eur., CVMP) or 14 days (9CFR). The cells are subcultured by preparation of freeze/thawed extracts with inoculation onto fresh monolayers of corresponding cell type (Ph. Eur., CVMP) or by at least one subculture (9CFR). The inoculated cultures are observed regularly for evidence of cytopathic agents. After at least 28 d.p.i. (Ph. Eur., CVMP) or 14 d.p.i. (9CFR), monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests (see Section 3 for details on EU and US differences). 2.3. Substances of animal origin Substances of animal origin may be used during the manufacture of veterinary vaccines such as serum and trypsin used in the
cultivation of cell seeds. These raw materials must be tested for freedom from extraneous agents once they have been prepared in an appropriate manner for inoculation onto detector cell cultures. For the detection of extraneous viruses, test substances must be suitably prepared. Solids are dissolved or resuspended in a suitable medium to create a solution or suspension containing at least 300 g/L of the test material (Ph. Eur., CVMP) or 3.75 ml or 15% of the ingredient in growth medium (9CFR). The test material is inoculated onto monolayers of at least 70 cm2 (Ph. Eur., CVMP) or 75 cm2 (9CFR) of suitable detector cells. The inoculated cells are cultured for a total of at least 21 days (Ph. Eur., CVMP and 9CFR) with regular observations for cytopathic agents (Fig. 4). At the end of each 7 day period, monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests. The remaining cells are subcultured (Ph. Eur., CVMP). Alternatively, cells are subcultured at least two times over the 21 day period, at the end of which monolayers of specified surface area are tested for cytopathic agents, by staining; haemadsorbing agents, by HA; and specified viruses, by IFA or similar specific tests (9CFR). In the EU, requirements for testing bovine serum used in the production of veterinary vaccines is outlined in CVMP guidance on “Requirements and controls applied to bovine serum used in the production of immunological veterinary medicinal products” [8]. General and specific tests should be performed prior to any serum inactivation treatment and further tests performed postinactivation. The general and specific tests applied must be capable of detecting specific viruses listed within the guidance document.
S. Sheridan, J. Coughlin / Biologicals 38 (2010) 340e345
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Fig. 3. Comparison of EU and US methods for extraneous agent testing of virus seeds. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
3. Endpoint tests 3.1. Staining for cytopathic effects Cells infected with cytopathic viruses display CPE or morphological changes. Cells exhibiting these changes can be fixed and stained with an appropriate cytological stain such as Giemsa and hematoxylin and eosin (H&E). In accordance with EU and US regulations, at least two monolayers of 6 cm2 (Ph. Eur., CVMP) or at least one monolayer of 6 cm2 (9CFR) of cells are stained. Cells are observed for inclusion bodies, giant cells or other abnormalities or lesions indicative of changes in cellular morphology which may be attributable to a viral contaminant. 3.2. Haemadsorption assay Some members of the virus groups of Orthomyxo-, Toga-, Parvoand Paramyxo-viruses may not always produce an obvious CPE in susceptible cell cultures. These viruses may be detected by a haemadsorption assay that utilises their haemagglutinin expression. Monolayers of at least 70 cm2 (Ph. Eur., CVMP) or at least one monolayer of 6 cm2 (9CFR) of cells are required. A suspension (0.2% as per 9CFR) of red blood cells including chicken, guinea pig and any species of concern is added to the test monolayer (human red blood cells may also be included). Cultures are incubated at 4 C, room temperature and 37 C (Ph. Eur., CVMP) or 4 C and
20e25 C (9CFR) for a period of 30 min and/or 60 min. The cultures are then washed and observed microscopically for evidence of haemadsorption. 3.3. Immunofluorescence assay Monoclonal or polyclonal antibodies specific for particular antigens are utilised to detect specific viruses in fixed cell cultures. The antibodies are labelled with a fluorescent dye and, when they are in contact with their specific antigen, the reaction site can be observed using a fluorescent microscope. If viral antigen is not present the antibodies are removed during the washing steps. This test is suitable for detecting non-cytopathic and non-haemadsorbing viruses. The specific viruses to be tested for by IFA are determined by the species of origin of the cell seed or virus seed under test and the intended species of the product. Monolayers of sufficient cells (Ph. Eur., CVMP) or at least 6 cm2 of cells under test (9CFR) are required. A positive control inoculated with 100e300 FAID50 (50% fluorescent antigen infectious doses) should be included and may be fixed for IFA prior to day 7 of the culture period if fluorescence is enhanced by doing so, provided that a test monolayer is also fixed at the same time, as well as at least 7 days after subculturing (9CFR). Regardless of the species of origin and target species, monolayers should be tested for Pestiviruses (Ph. Eur., CVMP) or bovine viral diarrhoea virus, reovirus and rabies virus (9CFR).
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Fig. 4. Comparison of EU and US methods for extraneous agent testing of substances of animal origin. WCS, working cell seed; MCS, master cell seed; HA, haemadsorption assay; IFA, immunofluorescence assay.
4. Avian vaccines Virus and cell seeds used in the production of veterinary vaccines for avian species must be tested to preclude extraneous agents. For the detection of avian derived contaminants, in vivo and egg tests are recommended in addition to in vitro assays using suitably sensitive avian detector cell lines. A full range of assays to detect avian viral agents would include the following tests: extraneous viruses using fertilized eggs (Ph. Eur., CVMP, 9CFR); extraneous viruses using cell cultures (Ph. Eur., CVMP); avian leucosis viruses (Ph. Eur., CVMP, 9CFR); avian reticuloendotheliosis virus (Ph. Eur., CVMP, Veterinary Services (VS) Memorandum 800.88); extraneous agents using chicks (Ph. Eur., CVMP, 9CFR); and chick anaemia virus (Ph. Eur., CVMP, VS Memorandum 800.89). Endpoint tests are based on observations for abnormalities and mortality (in vivo or egg tests) or CPE and HA (in vitro tests). Specific tests such as IFA, enzyme-linked immunosorbent assay (ELISA) or the complement fixation test for avian leucosis (COFAL) for viruses and other pathogens associated with avian species (e.g. chicken, duck, turkey or goose) are typically performed as endpoint tests for either the in vitro or in vivo assays [4].
As for mammalian virus seeds, neutralisation of the virus may be required for testing avian virus seeds for extraneous viruses in the various test systems prescribed. Polyclonal or monoclonal antibody is produced, as outlined for mammalian vaccines. The neutralising antiserum must be shown to be free from antibodies against, and free from inhibitory effects on, agents listed within the relevant regulations and guidelines [1,4]. Monospecific antisera for virus neutralisation are not required to be tested for antibodies against all of these viruses, if it can be shown that the immunizing antigen could not have been contaminated with antigens derived from the virus in question and if the virus is not known to infect the species of origin of the serum. Also it is not necessary to retest sera obtained from birds from specific pathogen free (SPF) chicken flocks (Ph. Eur). Batches of serum must not be prepared from any passage level derived from the virus isolate used to prepare the master seed lot or from an isolate cultured in the same cell line. The antiserum should be used minimally to neutralise (if possible) at least the virus content of 10 doses of vaccine per 0.1 ml (in vitro tests) or 0.2 ml (in vivo tests). Alternative methods for neutralising or removing the test virus may be used in the absence of a suitable neutralising antiserum.
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5. Fish vaccines
Acknowledgments
Virus and cell seeds used in the production of veterinary vaccines intended for fish must also be tested to preclude extraneous agents. Guidance provided by the CVMP recommends that testing be conducted as described for starting materials of mammalian origin. Endpoint tests using specific methods such as IFA should be included for the detection of pathogens of piscine origin [5].
We thank Doris Barnett, BioReliance Corporation, Rockville, Maryland, USA and Donna Gatewood, D.V.M., M.S, Center for Veterinary Biologics, Ames, Iowa, USA.
6. Discussion There are differences between EU and US requirements for extraneous agent testing of starting materials used in the production of veterinary vaccines. There are differences with respect to what stage starting materials should be tested at, as well as the specific methods that should be employed for these tests. In addressing concerns about extraneous agents for specific species, further differences exist; under EU requirements, lists of viruses that should be tested for by both general and specific methods are included whereas under US requirements, only viruses for which specific detection methods are required are listed. Furthermore, the species for which such lists are included are not the same; EU requirements include lists of extraneous agents of rodent, lagomorph and fish origin that are not included in US requirements (although there could be a requirement under US regulations to test for extraneous agents of these species if involved). Where lists for the same species do exist in EU and US regulations and guidelines, the viruses contained in these lists are not identical.
References [1] European Pharmacopoeia. In: Methods of analysis. 6th ed. Strasbourg: Council of Europe; 2009. [2] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality. vol. 7B, General requirements for the production and control of live mammalian bacterial and viral vaccines for veterinary use; March 1992 [7BIm1a (III/3182/91-EN)]. [3] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality. vol. 7B, General requirements for the production and control of inactivated mammalian bacterial and viral vaccines for veterinary use. ; March 1992 [7BIm1b (III/3181/ 91-EN)]. [4] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality, vol. 7B. Specific requirements for the production and control of avian live and inactivated viral and bacterial vaccines; November 1992 [7BIm3a (III/3363/92-EN)]. [5] Specific requirements for the production and control of live and inactivated vaccines intended for fish; September 1994 [7BIm9a (III/3590/92-EN)]. [6] Code of federal regulations title 9. Animals and animal products, Part 113 standard requirements; January 2009. [7] The rules governing medicinal products in the European Union. Guidelines, veterinary medicinal products, immunologicals, quality, vol. 7B, Table of extraneous agents to be tested for in relation to the general and species specific guidelines on production and control of mammalian veterinary vaccines; September 1994 [7BIm10a]. [8] Revised guideline on requirements and controls applied to bovine serum used in the production of immunological veterinary medicinal products. EMEA/ CVMP/743/00-Rev. 2; November 2005.