Brussels, 18 April, 2019

TRADE-EU-US-REG-COOP-CONSULTATION@ec.europa.eu

Subject: Call for proposal for regulatory cooperation activities.

Dear European Commission,

Introduction

The European Serum Products Association, ESPA, represents producers of animal sera, which are used in in-vitro cell culture. This technology has been widely used for over 60 year in the discovery, development and production of diagnostics, as well as life saving human and animal pharmaceuticals and therapies, while at the same time drastically reducing the use of lab animals.

The main application areas for cell culture include: academic and industrial research, industrial production of in-vitro diagnostics, human and animal vaccines, synthetic proteins such as monoclonal antibodies (mABs), stem cell therapy, gene therapy, immune therapy, and regenerative medicine. Most recently synthetic meat has been added to this list.

Cell culture media include 5-10 % sterile filtered serum, mostly foetal bovine serum (FBS), collected in slaughterhouses. While serum is used in the production process of therapeutics, purification processes eliminate the animal material from the final product.

Price differences between geographic origins create a fertile ground for misrepresentation and adulteration of FBS, which may influence the outcome of research projects and diagnostics. Many such cases have been identified and reported to affected victims.

With harmonization of the international trade of animal sera, price differences between origins will be reduced, along with the economic incentives for illegal activities. While FBS sourcing today covers most areas where cattle are harvested, price differences between origins have remained astronomical. Importation rules have been harmonized by most countries, following OIE guidelines, with the exception of the USA. The USA’s outdated and unjustified importation rules cause price differences and represent a barrier to trade and the development of the life science industry and research activities worldwide. It should be noted that USDA is aware their import requirements for animal serum are outdated. Two attempts were made by USDA in 19941 and 20062 to update and harmonize the import requirements with the OIE standards. However, because of opposition from US stakeholders, USDA ended up withdrawing both proposals.

1. Federal Register Vol. 59 No. 38, 25 February 1994. Proposed Rule-Importation of Fetal BovineSerum. https://www.govinfo.gov/content/pkg/FR-1994-02-25/html/94-4326 htm2 U.S. Animal Health Association (USAHA) 2007 Resolution No. 65 Importation of Fetal BovineSerum – USDA Response. http://www.usaha.org/upload/Resolution/resolution65-2007.pdf

[All redactions in the document are applied in accordance with Art.4.1(b)]

Proposal

ESPA therefore proposes the harmonization of regulations between the EU and the USA, covering trade and the use of animal serum, following standards set by World Animal Health Organization (OIE), World Health Organization (WHO), and World Trade Organization (WTO).

Advantages of harmonised trade rules for serum:

1. The limited availability of FBS is driving up the cost for the end-user. At the same time,

the global demand for FBS is increasing rapidly. While the EU is allowing the

importation of FBS from countries free from FMD, free with vaccination, and free

without vaccination, the US restricts serum imports to countries free from FMD

without vaccination. European researchers pay an average of ≤ 200 USD/litre for

FBS, whereas US researchers in some cases pay up to 1000 USD / litre due to the

limited supply.

2. The price difference between FBS from different geographical areas creates an

incentive for illegal activities. Many cases of misrepresentation have been identified

over the years. Raw material from lower priced areas is illegally labelled and sold as

a product from a higher priced geographical area.

3. Harmonized rules allow the free and open exchange of products, professional

information and services within industry and scientific sectors worldwide, and ensure

that markets for serum products operate in compliance with all applicable national and

international rules and regulations.

4. EU legislation, EMA guidance and the European Pharmacopeia3 provide an ample

supply of safe product. If harmonization is achieved by the US adjusting to EU rules,

the US end-user will have access to higher volumes of high-quality sera at lower price

level. In the opposite case of the EU copying the US import rules4, all FBS prices

worldwide would explode.

The truth about geographical origin

Some origins of serum are advertised as better or safer than others. This is a misleading marketing trick. Origin of raw material is defined as the abattoir where blood is collected. Where live cattle are crossing borders (as in North America and the EU), this definition of origin is pointless since it is not an accurate indication of the health status of the location where cattle were born and raised. After a raw product is imported for processing and finishing, the safety and quality of the product become the responsibility of the importer. When the finished product is ready for marketing and export, the country where processing occurred becomes the “origin”, as defined by the WTO5. This criteria is how the EU and US deal with Mexican FBS processed in the USA and exported to the EU, and how the US and New Zealand deal with Australian FBS processed in New Zealand.

3 The importation of animal sera into the EU is regulated by EC1069/2009, EC142/2011 and EC219/319. The use of bovine serum in production of pharmaceuticals is subject to EP2262 and EMA guidelines EMA-CHMP-BWP-457920-2012-R1 – (Human) and EMEA-CVMP-743-00-Rev.2 – (Veterinary) 4 The importation of animal sera into the USA is regulated by 9 CFR 94, 9 CFR 113, and USDA Veterinary Services Notice 98-05, Ruminant Serum Importation Requirements (1998). 5 WTO (1994) Rules of Origin, Article 3(b) “the country where the last substantial transformation (of the product) has been carried out”.

In terms of diseases, there are several aspects.

1. Bovine Spongiform Encephalopathy (BSE) We refer to the attached article, written byPercy Hawkes – Fetal bovine serum: geographical origin and regulatory relevance ofviral contamination – Hawkes. Bioresourc.Bioprocess. (2015) 2:34 DOI10.1186/s40643-015-0063-7: “However, as knowledge of BSE advanced, scientistsand government regulators from the World Organisation for Animal Health (OIE), theEuropean Union (EU) and the United States Department of Agriculture (USDA)determined that BSE is not transmitted in bovine blood products, whenappropriate slaughter practices are adhered to.”

2. Viral safety. We refer to the same article: “Regardless of geographical location, FBSfrom all 30 countries need to be tested and treated for the presence of multiple viruses.No one continent or country which produces FBS seems to have a real advantageover the others, since all countries have viruses of regulatory concern needing to betested for and eliminated in FBS. Based on this reality, there should be no risk for anycountry by adapting harmonized import rules based on the internationally acceptedprinciples.”

3. Irradiation to optimise safety for production of therapeutics: We refer to the article:

Gamma Irradiation of Animal Serum. Validation of Efficacy for Pathogen Reductionand Assessment of Impacts on Serum Performance, by Mark Plavsic, Raymond Nims,Marc Wintgens, and Rosemary Versteegen. Summer 2016 BioProcessing Journal[Vol.15/No.2]: The article describes the efficiency of irradiation for the reduction ofadventitious agents potentially present in serum. It explains the logic behind thestrategy that serum should be irradiated for the production of therapeutics.Conclusion: When irradiating animal sera for use in industrial processes, geographicalorigin becomes irrelevant from a safety point of view.

4. Regionalisation & testing: The lack of regionalisation in some geographical areas ofthe world, can compromise the exportability of serum from an entire country, in caseof a disease outbreak. ESPA strongly recommends that the EU and US authorities,consider accepting a negative test of representative serum samples, as an exportcriterium, in case of such a disease outbreak or as an alternative to the current healthstatements on the certificates.

In summary

ESPA proposes the harmonization of the import-export trade rules for animal serafor cell culture. This would increase the availability of sera, resulting in moreacceptable prices, while maintaining highest safety levels.

ESPA recommends that the requirements for bovine serum as described in theUSP and the EP, be aligned.

ESPA requests that the US and the EU accept in-vitro testing as an option andalternative to health statements on export-import certificates.

ESPA requests that both the EU and the US, actively encourage the use of 25 or30 kGy irradiation (depending on the application) to treat animal sera, regardlessof origin.

ESPA encourages the EU and the USDA to strengthen rules and regulatorycontrols over the animal origin ingredients used in the life science research, similarto regulatory controls over ingredients used in the pharmaceutical and foodindustries.

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serum media and has the advantage of very low levels of antibodies. On the other hand, it has the disadvantage of high costs and the need for testing and the elimination of adventitious viruses, as do other media of animal origin. Serum-free media have been developed to avoid the use of animals for cell culture media, and have met with great success, especially in the production of some proteins for medical uses (Cruz et al. 1998). However, pharmaceutical companies, diagnostic labs and researchers still depend heavily on FBS for most of their cell culture needs. Researchers have not been able yet to replicate all the growth factors present in FBS or to produce serum-free media on a large enough scale, allowing for a complete replacement of FBS. The pharmaceutical and biologics community will most likely continue depending on FBS for many more years.

ReviewOrigins, diseases and rule settingNot all countries qualify for exporting FBS because of certain diseases in their cattle populations, and the related import restrictions imposed by importing coun-tries. These restrictions are imposed because of the ani-mal diseases present in the exporting country and the perceived or real risk of viruses being present in imported FBS. The purpose of import requirements is to guarantee the absence of viruses of concern, by either prohibiting importation, or by other measures such as safety testing, and gamma irradiation. Once having passed importation requirements, safety testing and/or sterilization treat-ment, the imported FBS is considered to be free of all viruses of importation concern, and comparable to FBS from any approved origin.

Given the fact that the two largest global markets for animal-derived products are the United States of Amer-ica (USA) and Europe, the import requirements from the USDA and the European Commission (EC), to a great extent, have become the veterinary control standards for the FBS industry. Since the 1980’s, the principal source countries for FBS are from North, Central and South America, and Oceania. Europe is being added to this list, now that Bovine Spongiform Encephalopathy (BSE) is no longer considered to be transmitted by blood and blood products. The 30 countries where FBS is collected all have in common their freedom from the major con-tagious viral diseases of cattle, such as Foot and Mouth Disease (FMD), Rinderpest, Peste des petits ruminants, and Rift Valley Fever.

The cattle diseases of concern for FBS are those which cross the placental barrier of the cow and infect the calf fetus, thus contaminating FBS and making it unsuit-able for use in cell cultures. From a geographic perspec-tive, some of these viruses (adventitious viruses) are

considered to have a worldwide distribution, and others (viruses of importation concern) are limited to certain regions of the world. Table 1 compares the disease status of the 30 FBS exporting countries for eight adventitious viruses and for six viruses of importation concern. The sources for this information are the OIE, the USDA, and the EU.

Adventitious virusesThe USDA (USDA 9 CFR 113.46-53) and EU (EMEA-CPMP-BWP-1793-02) regulations require that all FBS, regardless of country of origin, be tested and/or treated (by heat or gamma irradiation) to assure its freedom of the following eight adventitious viruses: bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), parainfluenza 3 (PI3), rabies, reovirus 3 (REO3), bovine adenovirus (BAV), bovine parvovirus (BPV), and bovine respiratory syncytial virus (BRSV). Because these eight viruses affect cattle in all continents of the world, they may unintentionally be present in FBS from any origin. The cell culture testing procedures required by USDA and EU serve not only to detect these eight viruses, but also to detect hemagglutination/hemadsorption and cytopathic effects caused by other viruses, which can contaminate FBS.

Even though these eight adventitious viruses are consid-ered to be present in all cattle producing areas, the OIE (http://www.oie.int/animal-health-in-the-world/official-disease-status/fmd/) reports the following exceptions:

Bovine Viral Diarrhea (BVD) is reported absent in three Scandinavian countries (Finland, Norway and Sweden). Several other European countries have also achieved significant success toward eradicating BVD (Switzerland, Austria, Scotland, Ireland, Sweden, Den-mark and Germany).

Infectious Bovine Rhinotracheitis (IBR) is also reported absent in 4 Scandinavian countries, the same three coun-tries mentioned above, plus Denmark.

Rabies has never been reported in New Zealand, and is reported absent in Australia, as well as the four Scandina-vian countries mentioned above, plus Belgium, Germany and Ireland.

Viruses of importation concernThe viruses of concern for FBS which do not have a worldwide distribution are also of concern when import-ing FBS. Regulations from USDA (Veterinary Services Notice 1998) and EU (Regulation EC No 294-2013) identify six viruses of importation concern for the FBS producing areas of the world: Foot and Mouth Disease (FMD), Vesicular Stomatitis, Blue Tongue, Akabane, Aino (Veterinary Services Notice 1992), and Schmallen-berg (USDA Schmallenberg Restrictions). FMD is the

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Table 1 Regulatory diseases of concern for Fetal Bovine Serum—comparison of animal health status of countries of FBS origin

FBS exporting countries

Adventitious viruses of concern

Considered worldwide distribution by USDA and EU Source: 2013 OIE data Total adventitious virusesParainfluenza

3Reovirus 3 Bovines

adenovirusBovine parvovirus

Bovine respiratory syncytial virus

Bovine viral diarrhea (BVD)

Infectious bovine rhinotrachei-tis.(IBR)

Rabies

Finland + + + + + 2010 1994 2007 5

Norway + + + + + 2005 1992 2011 5

Sweden + + + + + 2011 1995 1886 5

Denmark + + + + + + 2005 2002 6

New Zealand + + + + + + + – 7

Belgium + + + + + + + 2008 7

Chile + + + + + + + + 8

Germany + + + + + + + 2005 7

Ireland + + + + + + + 1903 7

Uruguay + + + + + + + + 8

Argentina + + + + + + + + 8

Canada + + + + + + + + 8

Colombia + + + + + + + + 8

Dominican Republic

+ + + + + + + + 8

El Salvador + + + + + + + + 8

Guatemala + + + + + + + + 8

Honduras + + + + + + + + 8

Holland + + + + + + + + 8

Mexico + + + + + + + + 8

Nicaragua + + + + + + + + 8

Panama + + + + + 2007 + + 7

Paraguay + + + + + + + + 8

Peru + + + + + + + + 8

Poland + + + + + + + + 8

Australia + + + + + + + 1867 7

Brazil + + + + + + + + 8

Costa Rica + + + + + + + + 8

France + + + + + + + + 8

Spain + + + + + + + + 8

United States + + + + + + + + 8

FBS exporting countries

Viruses of importation concern Total viruses of FBS concernSource: 2013 data from OIE, USDA and EU Total viruses

of import concernFoot and

mouth disease (FMD)

Vesicular stomatitis (VS)

Bluetongue (BT)

Akabane Aino virus

Schmallenberg virus

Finland 1959 – – – – + 1 6

Norway 1952 – 2010 – – + 1 6

Sweden 1966 – 2009 – – + 1 6

Denmark 1983 – 2009 – – + 1 7

New Zealand – – – – – – 0 7

Belgium 1976 – 2008 – – + 1 8

Chile 1987 – – – – – 0 8

Page 4 of 5Hawkes. Bioresour. Bioprocess. (2015) 2:34

only one of these diseases which is not an insect vectored disease.

Non‑insect vectored diseaseFoot and Mouth Disease (FMD) All 30 countries are officially recognized by the OIE as free of FMD (http://www.oie.int/animal-health-in-the-world/official-disease-status/fmd/). The South American countries listed in Table  1 are free of FMD with vaccination (except Chile which is free without vaccination), and the remaining countries on the list are free of FMD without vaccination.

Currently, the major difference between US and EU importation rules for FBS is over the definition of FMD free status. The EU accepts FBS from countries free of FMD, both with and without vaccination, whereas the USA only accepts FBS from countries free of FMD with-out vaccination. As a result, today’s commercialization of

FBS from South American origins (with the exception of Chile) is limited to Europe and Asia.

Even with the conservative approach taken by USDA, it should be noted that there are no literature reports of the FMD virus or FMD antibodies ever being found in bovine fetuses or FBS.

Insect vectored diseasesVesicular stomatitis virus (VSV) is of concern to the European countries because the virus only exists in the Americas and because its clinical presentation is identi-cal to FMD, even though the two viruses are from differ-ent families (VSV from the Rhabdoviridae and FMD from Picornaviridae). VSV is spread by insects and is endemic in the tropical and semitropical areas of the Americas. Every few years, VS spreads into the adjacent temperate areas of North and South America.

(Year) indicates year disease last reported; (+) disease is present; (−) disease has never been reported.

Sources: OIE Animal Health Status; USDA 9 CFR 113.46-53; EMEA-CPMP-BWP-1793-02.

Table 1 continued

FBS exporting countries

Viruses of importation concern Total viruses of FBS concernSource: 2013 data from OIE, USDA and EU Total viruses

of import concernFoot and

mouth disease (FMD)

Vesicular stomatitis (VS)

Bluetongue (BT)

Akabane Aino virus

Schmallenberg virus

Germany 1988 – 2009 – – + 1 8

Ireland 2001 – – – – + 1 8

Uruguay 2001 – – – – – 0 8

Argentina 2006 1986 + – – – 1 9

Canada 1952 1949 + – – – 1 9

Colombia 2009 + 2007 – – – 1 9

Dominican Republic

– – + – – – 1 9

El Salvador – + 1997 – – – 1 9

Guatemala – + 1998 – – – 1 9

Honduras – + 2004 – – – 1 9

Holland 2001 – 2009 – – + 1 9

Mexico 1954 + 2010 – – – 1 9

Nicaragua – + 2009 – – – 1 9

Panama – + No Info – – – 2 9

Paraguay 2012 – Unknown – – – 1 9

Peru 2004 + 2004 – – – 1 9

Poland 1971 – – – – + 1 9

Australia 1871 – + + + – 3 10

Brazil 2006 + + – – – 2 10

Costa Rica – + + – – – 2 10

France 2001 – + – – + 2 10

Spain 1986 – + – – + 2 10

USA 1929 + + – – – 2 10

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Blue tongue virus (BTV) occurs in all tropical and semi-tropical climates of the world where biting midge vectors exist, and there are 24 distinct serotypes in different areas of the world (http://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_cards/BLUETONGUE.pdf). Importation requirements are in place for this virus, in order to prevent the introduction of new serotypes from other parts of the world. Every few years, the virus reappears in areas where biting midges exist. USDA requires that serum from all countries, except Canada and New Zealand, be tested for BTV. Canada has the same serotypes of BTV as the USA, and New Zealand has never reported BTV.

Akabane, aino and schmallenberg viruses (Simbu Serogroup viruses) are transmitted by insects and cause deformities and death in bovine fetuses. USDA requires Australian serum be tested for Akabane. The same test detects the Aino Virus, which is also present in Australia. USDA also has import restrictions for European animal byproducts relating to Schmallenberg.

Comparing origins of FBSFirst and foremost, it should be noted that all 30 coun-tries listed on Table  1 are officially recognized by the OIE as being free of FMD, a remarkable accomplishment requiring excellent disease detection and surveillance programs.

Regarding the other five viruses of importation con-cern: all 30 countries are free of Akabane and Aino viruses, except Australia; only the European countries are affected by the Schmallenberg virus; only the Americas are affected by VSV; and all three continents represented are occasionally affected by different strains of BTV.

Next, regarding the eight adventitious viruses: all 30 countries are considered to be infected with PI3, REO3, BAV, BPV and BRSV; six European countries, as well as Oceania are free of Rabies; and several Scandinavian countries report the absence of BVD and IBR.

The countries reporting the fewest (6–7) viruses of concern for FBS are Finland, Norway, Sweden, Denmark and New Zealand.

The countries reporting the most (10) viruses of con-cern for FBS are Australia, Brazil, Costa Rica, France, Spain, and the USA.

ConclusionsRegardless of geographical location, FBS from all 30 countries needs to be tested and treated for the presence of multiple viruses. No one continent or country which

produces FBS seems to have a real advantage over the others, since all countries have viruses of regulatory con-cern needing to be tested for and eliminated in FBS.

The comparisons made in this article, concerning the animal health statuses of the major FBS producing countries, show why a correlation between geographi-cal origin and the “quality” of FBS based on the country’s animal health status is not reasonable.

Just because the prices of FBS from certain origins might be higher, does not mean that those origins are “safer”, in terms of the number of viruses needing to be tested for and eliminated.

Once having passed importation requirements, safety testing and sterilization treatment, FBS is considered to be free of all viruses of importation concern, and compa-rable to FBS from any other approved origin.

Finally, safe FBS can come from any of the USDA and EU approved origins.

AbbreviationsBAV: bovine adenovirus; BPV: bovine parvovirus; BRSV: bovine respiratory syncytial virus; BSE: bovine spongiform encephalopathy; BTV: blue tongue virus; BVD: bovine viral diarrhea; EC: European Commission; EMEA: Euro-pean Medicines Agency; EU: European Union; FBS: fetal bovine serum; FMD: foot and mouth disease; IBR: infectious bovine rhinotracheitis; OIE: World Organization for Animal Health; PI3: parainfluenza 3; REO3: reovirus 3; USA: United States of America; USDA: United States Department of Agriculture; VSV: vesicular stomatitis virus.

Competing interestsThe author has been doing consulting work for Biowest (http://www.biowest.net/) for the last 10 years. This article has been written as part of the author’s fee basis work for Biowest. The author declares that he has no competing interests.

Received: 21 April 2015 Accepted: 13 July 2015

ReferencesCruz H, Moreira J, Stacey G, Dias E, Hayes K, Looby D et al (1998) Adaptation

of BHK cells producing a recombinant protein to serum-free media and protein-free medium. Cytotechnology. doi:10.1023/A:1007951813755

Davis D, Drake Hirschi S (2014) Fetal bovine serum: what you should ask your supplier and why. BioProcess J 13(1):19–21. http://dx.doi.org/10.12665/J131.DavisHirschi

Siegel W, Foster L (2013) Fetal bovine serum: the impact of geography. Bio-Process J 12(3):28–30. http://dx.doi.org/10.12665/J123.Siegel

Veterinary Services Notice (1992) Ruminant serum (RS) import requirements, USDA-APHIS, October 29, 1992

Veterinary Services Notice 98-05 (1998) Ruminant serum import requirements, USDA-APHIS, March 19, 1998

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Gamma Irradiation of Animal Serum: Validation of Efficacy for Pathogen Reduction and Assessment of Impacts on Serum Performance

of appropriate viruses is based on several factors such as:

• a risk analysis for the serum product and the serummanufacturing process;

• the epizootic status (i.e., animal disease epidemiology)of the serum’s country of origin;

• applicable information that might be provided by theOffice International des Epizooties (OIE);

• consideration of new and emerging pathogens;

• the potential risk to the serum end-user;

• the potential for adventitious agents in the final medic-inal product manufactured using the serum; and

• the safety risk to patients —recipients of the medicinalproduct manufactured using the serum.

As a standard practice, the panel of challenge viruses selected should represent:

• different families including both RNA and DNAviruses possessing diverse genomic organ izations(e.g., single-stranded [ss] and double-stranded [ds],negative- and positive-sense, linear, circular,segmented, and non- segmented);

• viruses with and without envelopes;

• varied particle sizes ranging from small to large; and

• agents of differing levels of resistance to gammairradiation.

The spiking viruses should be of known history and confirmed identity, high quality, adequate purity, and at sufficiently high titer (preferably >106 tissue culture infec-tive dose50 [TCID50] or plaque-forming units [PFU] per mL) in order to be capable of determining the actual upper limits of virus titer reduction with reasonable certainty.

Care should be taken regarding the use of field isolates vs. laboratory-adapted virus strains. The field strains may exhibit different characteristics (more quasi-species, diverse populations, defective and/or empty particles) and behaviors (different growth profiles in cell culture and sensitivities to inactivation) than those of laboratory- adapted strains. It is advisable to use laboratory virus stocks that have as many similarities as possible to the field isolates, with a low number of passages from isolation, if possible.

Test Matrix ConsiderationsIt has been well-established that serum products, as well

as other types of animal-derived materials, display various degrees of lot-to-lot (inter-lot) variability. This variability is inherent to serum composition (matrix) which, in turn, may affect the efficacy of gamma irradiation for inactivation of viruses obtained in the study. For example, serum may contain antibodies or other non-antibody inhibitors that may specifically or non-specifically bind with or neutralize spiking viruses. The presence of such factors in the serum may mask the presence of the spiking viruses, leading to

underestimates of their titers using infectivity assays. Such adverse impacts on the virus titration assays used prior to and following irradiation can impact the overall irradiation study interpretation. Therefore, careful selection of repre-sentative serum for validation of the gamma irradiation process is very important.

The serum should be tested for the presence of anti-viral antibodies, and only antibody-free serum should be used in the validation study. Preferably, three lots of serum should be used for virus spiking purposes in order to better assess the impact of inter-lot variability. The antibody-free serum matrix should be tested for potential interference and cyto-toxicity prior to conducting the inactivation study itself in order to determine any virus-blocking effects (interference) or potential toxic effects of the matrix on the indicator cells (cytotoxicity). Serum determined to be free of such interfer-ence or cytotoxic effects may be used for the actual virus spiking and irradiation study. This approach to mitigating the adverse impacts of neutralizing antibodies is consid-ered more appropriate than the use of proxy serum derived from other animal species (e.g., the use of horse serum for spiking studies involving BVDV).

Where possible, a worst-case matrix should be deter-mined. This may be defined as serum that is free of cross-reacting antibodies and other interfering molecules with a relatively high total protein, lipid, and/or hemoglobin content. Such a matrix could, under certain conditions, be protective of the spiking viruses, impact virus dispersion and homogeneity within the serum being irradiated, or increase viral aggregation. Each of these factors may limit, to some extent, the determined efficacy of viral inactiva-tion by gamma irradiation.

Appropriateness of the Scaled-Down ModelValidation of viral inactivation is not typically performed

at the commercial serum lot scale. Therefore, a scaled-down process is utilized for virus spiking purposes during the validation. The down-scale model should be carefully designed to best represent an actual large-scale irradi-ation process in terms of serum type, matrix, packaging features, shipping provisions, dose-mapping, and irradi-ation process.

Matrix Spiking and Study ControlsOnce an acceptable serum matrix has been selected, the

spiking virus stocks have been prepared, a scaled-down model is established, and the possibility of interference and cytotoxicity has been ruled out, the experiment itself may be conducted. Virus is spiked (<10% vol/vol) into serum bottles (containers) under aseptic conditions such that one bottle contains one spiking virus type. The serum-virus mixture should be mixed properly to ensure homogenous virus distribution and to minimize the occurrence of virus aggregates. The spiking is then repeated with each virus type until three different serum lots have been spiked with each of the panel of selected viruses.

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Gamma Irradiation of Animal Serum: Validation of Efficacy for Pathogen Reduction and Assessment of Impacts on Serum Performance

The following controls are typically incorporated into the experimental design: • negative (non-inoculated) serum;• spiked serum shipped to the irradiation facility but not

irradiated;• spiked stability (hold), kept frozen in the laboratory;• virus stability (hold), kept frozen in the laboratory; and• other controls as appropriate.

Establishing and Documenting Dosimetry In order to accurately monitor the applied irradiation

dose (especially product-absorbed dose), multiple dosim-eters may be placed at appropriate points within the packaging configuration. Dosimeters can be immersed inside bottles of serum before freezing, affixed to the exte-rior bottle surfaces at several locations, and/or attached to the outside of cases of bottles in various spots. The irra-diation containers (e.g., totes, pallets, carriers) should be representative of commercial applications in terms of size, type, and packing density. However, it is recognized that the bottles used in the down-scale inactivation studies may differ (e.g., volume and style) from those used for commer-cial-scale serum bottling and irradiation. Validation of dosimetry for an irradiation process will be addressed in a subsequent article in this series.

Determination of Inactivation per kGy Applied DoseIt is a regulatory requirement that the kinetics of inac-

tivation be evaluated during the validation of inactivation steps.[1, 3] Since gamma irradiation renders adventitious agents non-infectious by inactivating the genomic mate-rial, it is expected that the kinetics of inactivation will be evaluated. This is accomplished by irradiating the spiked serum with a series of increasing irradiation doses (e.g., 0–60 kGy in 5 or 10 kGy increments) using at least one bottle per serum lot, per virus, and per test dose.

The spiking virus titers are determined before and after irradiation for each dose, and the reduction of virus titer resulting from the irradiation is plotted vs. dose for each virus to establish the kinetics of virus inactivation for that virus. The resulting information is often expressed in terms of a D value (i.e., the dose in kGy required to cause a 1 log10 reduction in titer), although we find it more convenient to express the result in terms of the log10 inactivation obtained per kGy irradiation dose.[9]

Virus Quantitation ConsiderationsThe methods to be used for determining virus titer pre-

and post-irradiation are of critical importance in assessing the efficacy of gamma irradiation. Considering that gamma irradiation targets the viral genome, potentially causing strand breaks and other types of damage to the nucleic acids, it is essential not to use nucleic acid-based analytical tools (e.g., polymerase chain reaction [PCR]) for determining

virus titer. Although the virus may be completely inac-tivated (rendered non-infectious), nucleic acid testing methods might still be able to detect fragments of the viral nucleic acids, making it impossible to interpret the study results. Therefore, cell-based infectivity methods are used for virus titrations and virus reduction titers are analyzed using assays such as TCID50, PFU, fluorescent focus assay (FFA), etc. The methods should be validated at least as to specificity and sensitivity (limit of detection), and any method limitations should be well understood. Finally, the testing facility and its staff should be experienced and knowledgeable in the fields of virology, method execution, and data interpretation.

Data InterpretationData analysis and interpretation are typically performed

as described in ICH Q5A(R1)[1] and USP <1050.1>.[3]

Establishing the Irradiation Dose RangeThe applied dose (fluency) range to be used for irradi-

ating serum typically represents a window between the lower and higher irradiation dose, where sufficient virus inactivation is attained while the serum quality attributes remain minimally affected. An example of an irradiation dose range for treatment of serum products is 30–50 kGy. Although the lower value of the range (30 kGy) may repre-sent a minimum due to EMA[5] and Ph. Eur.[7] requirements, it can be set higher to assure greater virus reduction. The upper value of the range can also vary depending on the potential impact of higher doses on serum quality and performance.

The actual dose range applied during irradiation is process application-specific and various end-users may require slightly different ranges, considering empirically determined impacts on serum performance in their own process applications (detailed in section 3). In general, irradiation dose ranges that are overly narrow (e.g., <15 kGy difference between lower and upper values) can be diffi-cult to deliver by irradiators in a consistent manner, likely resulting in periodic deviations and potential product rejection. This topic will be addressed in greater detail in a subsequent article in this series.

The inactivation efficacy results that have been reported in the literature to date are described next.

2. Efficacy of Gamma Irradiation for Pathogen Reduction in Serum

As there have been no additions to the literature addressing efficacy of gamma irradiation for inactivating potential viral contaminants in animal serum since our previous review was published[9], we provide a summary of the most important findings of that review in this paper. In order to set the stage for the high-level discussion on efficacy presented below, it is necessary to discuss some of the mechanistic aspects of viral inactivation by gamma irradiation.

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Gamma Irradiation of Animal Serum: Validation of Efficacy for Pathogen Reduction and Assessment of Impacts on Serum Performance

° Revalidation of dosimetry after each replenishment of the source 60cobalt

° Dose-monitoring • Product appearance, both in the frozen and liquid

(thawed) state, looking particularly for:

° Color changes

° The presence of observable particular matter

° Stratification and homogeneity after bottle mixing • Serum filterability post-irradiation • Full biochemical profiling to include pH, osmo-

lality, and electrophoretic pattern (EPP) in order to understand specific changes in serum biochemistry (e.g., references[17, 18])

• Cell culture performance (e.g., references[10, 11, 17-20]) eval-uations using a variety of cell types and culture systems that are most often used by biopharmaceutical and vaccine manufacturers

° Cells should be maintained for at least 3–5 passages in medium supplemented with the irradiated serum.

° Cells should be monitored for performance alongside the same cells maintained on medium supplemented with non-irradiated serum of the same lot.

° Key performance indices should include, but not be limited to:

- Cell morphology - Cell density

- Doubling time - Metabolic profile

- Cell viability - Genetic toxicology

• Stability of the irradiated serum, employing appropriate stability-indicating assays

• Extractable and leachable studies on the final serum packaging container (used for commercial lots) exposed to irradiationA technical report summarizing this knowledge of the

gamma irradiation process and the comparability in serum characteristics should be prepared. This may then be freely shared with potential end-users of the irradiated serum. Although serum manufacturers are not responsible for the development of a comparability strategy to be employed by serum end-users, the serum provider’s report is typically requested by end-users to supplement their own compa-rability evaluations (see the next section).

Analysis of Irradiated Serum by Serum End-UsersTypically, end-users rely to some extent on the knowl-

edge and technical information provided to them by the serum producers. However, serum users following good manufacturing practices (GMP) are required (e.g., refer-ence[21]) to conduct additional testing and characterization when switching from non-irradiated to irradiated serum. The key question GMP serum users need to address also concerns comparability: What is the impact of the irradi-ated serum on the manufacturing process (cell culture and

purification) and the drug substance/drug product in compar-ison with the same process employing non-irradiated serum?

In order to address such a question, end-users of irradiated serum are expected to develop their own product- specific comparability strategies to satisfy regu-latory requirements extant across various world regions. In general, the following testing and characterization of more than one serum lot are typically considered:• Cell culture and purification performance testing using

representative small-scale models and, if appropriate, larger engineering-scale models employing various in-process controls

• Biologic drug substance (active pharmaceutical ingre-dient), and where applicable, product testing, and characterization

• Process validation at appropriate scale (e.g., GMP or other representative scale)

• Agency interactions and submission of comparability data for use of irradiated vs. non-irradiated serum

• Implementation in commercial manufacturingWhen irradiated serum is used in the early phase of

process development, the comparability testing exercise is not required. In this case, the analytical testing outlined above will be modified and built into the biologic drug substance process development. Therefore, the process development file should contain all relevant details on raw materials, cell culture, purification, and drug substance.

For end-users who are not manufacturing GMP prod-ucts, some form of comparability testing may still need to be done simply to confirm that responses obtained in their particular cell culture applications are not substantially different when using irradiated vs. non-irradiated serum. This possibility should be explored before switching to irradiated serum.

ConclusionsThis paper is part of a series of articles intended to

provide more transparency around the processes involved in the gamma irradiation of serum. The topical series has been introduced in a separate paper in this journal.[22]

Gamma irradiation of animal serum products is a mature barrier (risk mitigation) technology that has been utilized for several years. Numerous serum manufacturers now offer irradiated serum commercially after having evaluated and/or validated the efficacy of gamma irradiation and the potential impacts on serum performance. Many serum end-users have been relying on irradiated serum in their specific product manufacturing processes as a means of mitigating the risks of encountering adventitious microbial contaminants. This treatment modality has been proven to effectively inactivate mollicutes as well as a range of common animal viruses (e.g., bunyaviruses, retroviruses, herpesviruses, rhabdoviruses, flaviviruses, picornaviruses, and reoviruses). It has also been firmly established in various well-controlled studies that several virus groups

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Acknowledgement

(e.g., parvoviruses, polyomaviruses, and circoviruses) are more radio-resistant and are inactivated less effectively. For these reasons, gamma irradiation has been viewed as a means of “reducing” but not “eliminating” the viral risk associated with the use of serum in cell culture media.

While the serum industry evaluates biochemical, phys-icochemical, and performance variations post- irradiation, it is expected that end-users will develop their own product- specific compatibility and comparability strategies.

Validation of gamma irradiation is of paramount impor-tance as it ensures a consistent and well-controlled serum treatment process while providing consistent quality, safety, and performance for various end-user applications. Historically, gamma irradiation process development and validation has consisted of:

• Viral inactivation• Irradiated product evaluation• Optimal irradiation dose-range determination

• Dose-mapping

• Product packaging

• Product temperature maintenance

• Product shipment conditions

• The irradiation process itself

While many of these aspects should be performed for any new irradiation process, a case can be made that not all need to be repeated for changes to existing processes. Under certain circumstances (i.e., when the same types of viruses are spiked into the same types of matrices), and in agreement with end-users, the accumulated knowledge of virus susceptibility can be used to justify the evaluation of a more limited set of irradiation doses (e.g., 30–50 kGy). Such an irradiation window can then be used for conducting irra-diation process validation followed by thorough product characterization and performance evaluation in relevant application systems by the end-user.

Gamma Irradiation of Animal Serum: Validation of Efficacy for Pathogen Reduction and Assessment of Impacts on Serum Performance

The authors would like to acknowledge the following industry experts for their contributions: Randy Fitzgerald (Proliant), Andy Pratt (GE Healthcare), Bart Croonenborghs (Sterigenics), Robert J. Klostermann (Merial), Karl Hemmerich (Ageless Processing Technologies), Huw Hughes (Zoetis), Debbie Elms (Thermo Fisher Scientif ic), James Dunster (Moregate BioTech), and Sue Brown (TCS Biosciences). We are also grateful to Julia Hoffmann for her excellent administrative assistance.

[1] International Conference on Harmonisation. Topic Q5A(R1). Quality of biotechnological products: viral safet y evaluation of biotechnology products derived from cell lines of human or animal origin. September 23, 1999. http://www.ich.org/f ileadmin/Public_Web_Site/ICH_Products/Guidelines/Qualit y/Q5A _R1/Step4/Q5A _R1_ _Guideline.pdf[2] Food and Dr ug Adminis trat ion. Points to consider in the char-ac terization of cel l l ines used to produce biologicals. July 12, 1993. h t t p : / / w w w . f d a . g o v / d o w n l o a d s / B i o l o g i c s B l o o d V a c c i n e s /G u i d a n c e C o m p l i a n c e R e g u l a t o r y I n f o r m a t i o n /OtherRecommendationsforManufac turers/UCM062745.pdf[3] United States Pharmacopeia, USP General Chapter <1050.1>. Design, evaluation and characterization of viral clearance procedures.[4] United States Pharmacopeia, USP General Chapter <1024>. Bovine serum.[5] European Medicines Agency. EMA/CVMP/743/00-Rev.2. Requirements and controls applied to bovine serum used in the production of immunolog-ical veterinary medicinal products. November 9, 2005. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/10/WC500004575.pdf[6] European Medicines Agency. EMA/CHMP/BWP/457920/2012 Rev. 1. Guideline on the use of bovine serum in the manufacture of human biological medicinal products. May 30, 2013. http://www.ema.europa.eu/ema/pages/includes/document/open_document.jsp?webContentId=WC500143930[7] European Pharmacopoeia, Monograph: Bovine Serum, 01/2008: 2262, pp 1506–7. [8] World Health Organization. Recommendations for the evaluation

of animal cell cultures as substrates for the manufacture of biological medicinal produc ts and for the charac terization of cel l bank s, 2010. http://www.who.int/entity/biologicals/vaccines/TRS_978_Annex_3.pdf ?ua=1 [9] Nims RW, Gauvin G, Plavsic M. Gamma irradiation of animal sera for inac tivation of viruses and mollicutes – a review. Biologicals, 2011; 39(6):370 –7. ht tp://dx.doi.org/10.1016/j.biologicals.2011.05.003[10] Plavsic MZ, Daley JP, Danner DJ, Weppner DJ. Gamma irradiation of bovine sera. Dev Biol Stand, 1999; 99:95–109. PMid:10404881.[11] Purtle DR, Festen RM, Etchberger KJ, Caffrey MB, Doak JA. Validated gamma radiated serum products. Research report R013. SAFC Biosciences, 2006. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Produc t_Information_Sheet/r013.pdf[12] Wyat t DE, Keathley JD, Williams CM, Broce R. Is there life af ter irradiation? Par t 1: Inactivation of biological contaminants. BioPharm, 1993; 6(5):34–40.[13] Hansen G, Wilkinson R. Gamma radiation and virus inac tivation: new f indings and old theories. Art to Science, 1993; 12(2):1– 6.[14] House C, House JA, Yedloutschnig RJ. Inactivation of viral agents in bovine serum by gamma irradiation. Can J Microbiol, 1990; 36(10):737–40. PMid:2123735[15] Plavsic ZM, Bolin S. Resistance of porcine circovirus to gamma irradiation. BioPharm, 2001; 14(4):32– 6.[16] Gauvin G, Nims R. Gamma-irradiation of serum for the inac ti-vation of adventitious contaminants. PDA J Pharm Sci Technol, 2010; 64(5):432–5. PMid:21502047

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