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Safety testing of Biotechnology Products derived from Cell Lines of Human and Animal Origin

It was in 1982 that the World Health Organisation (WHO) introduced safety testing guidelines for the use of continuous cell lines employed as substrates for the production of inactivated viral vaccines. Since then, progress in the biomedical sciences and the exploitation of biotechnology has led to the development of new biological medicinal products at an unprecedented rate. Today's philosophy of the WHO, The Center for Biologics Evaluation and Research (CBER) at the US Food & Drug Administration (FDA), and the Japanese Ministry of Health for the regulation of biotechnology products, is a case by case approach based on risk- benefit assessments, sound scientific principles and the experience with these products in the last two decades. The safety testing guidelines issued by all these regulatory agencies and the major progress made recently by the International Conference on Harmonisation (ICH) are the basis for ensuring the safety of new biological entities.

Major concern: viral contamination

The major concern when using mammalian cell lines for production of a biotechnology product, is the risk of viral contamination. Such contamination could have serious clinical consequences and can arise from the contamination of the cell substrate or by the introduction of adventitious viruses during production. To date however, biotechnology products derived from cell lines have not been implicated in the transmission of viruses. To ensure the continued safety of these products, the adoption of a sound virus testing program is essential. The strategy that has evolved since 1982 consists of three complimentary approaches: selecting and testing cell lines and raw materials (including cell culture media and animal derived raw materials), for the absence of undesirable viruses which may be infectious and/or pathogenic for humans; assessing the capacity of the manufacturing process to inactivate or remove infectious viruses; testing the product at appropriate stages during production for the absence of contaminating infectious viruses. Confidence that infectious virus is absent from the final purified product cannot, in most instances, be derived solely from direct testing due to the inherent limitations of viral assays, that is, the ability to detect low levels of viral contamination depends for statistical reasons on the size of the sample being tested. Testing the capacity of the purification scheme to remove or inactivate known or unknown viruses is therefore a critical element in the overall safety testing strategy.

Potential sources of viral contamination

1. Contamination of the Cell Substrate. Viruses could be introduced into the cell substrate from several sources, including derivation from infected animals (e.g. rodent viruses in murine hybridomas and chinese hamster cells) and the use of contaminated cell culture reagents, such as bovine viruses from animal sera or porcine viruses from trypsin. Endegenous viruses, such as retroviruses, pose a particular problem in assessing the safety of cell substrates, as they may be transmitted in the germ line, since the viral genome persists within the cell. These endegenous retroviruses may be expressed without deleterious effects on the cells, and could be infectious to human cells. 2. Adventitious viruses introduced during the manufacturing process. The likely sources of contamination include the use of contaminated cell culture media, a breakdown in GMP allowing operator or other external contamination, the use of a contaminated excipient during formulation or other reagents used in the process, such as a monoclonal antibody affinity chromatography column. The importance of the sourcing and the qualification of all raw materials used in the manufacturing process has become a key area for the biopharmaceutical industry, not just to exclude viral contamination, but also to address the concerns of other contaminants such as transmissable spongiform encephalopathy agents, such as BSE. Cell culture media which exclude all animal components are now widely used in the industry to reduce the potential contamination problems.

Purified Bulk

The testing of the purified bulk will vary, depending upon the results of the cell bank and unprocessed bulk testing, however, the key assays to perform are: Microbiological contaminants: Sterility, mycoplasma Residual DNA: Host cell DNA assay

Final Filed Product

The tests to perform include: Microbiological contaminants: Sterility Endotoxin: LAL or Rabbit Pyrogen Toxicity: Abnormal Toxicity or General Safety

Viral Clearance Studies

These studies are conducted to assess the capacity of the purification process to remove or inactivate viruses or other contaminants. The type and extent of viral clearance studies to be conducted, will depend on various factors and should be considered on a case by case basis. The results of the cell bank testing, in process testing, the nature of the culture medium used, the product and its application and the ability of the process to inactivate or remove viruses, are all factors which influence the design of the study. When using a rodent cell line, for example, to manufacture a biopharmaceutical, the endogenous retroviruses present in the cells will require the use of a murine retrovirus in the viral clearance study, as well as considering the use of other relevant or model viruses.

Regulatory Guidelines

Please consult the Interesting Links pages and visit the websites of FDA, CPMP and ICH for documents.

Safety Testing Outline

The safety testing strategy should address all the following stages in the production of a biopharmaceutical:

Master Cell Bank

As all production lots will originate from the MCB, the safety testing is very comprehensive and includes the following type of assays. The origin and history of the cell line will determine the specific virological or microbiological assays required: Microbiological contaminants: Sterility, mycoplasma Cell Line Identification: Isoenzyme analysis or DNA fingerprinting or karyology Retroviruses: Reverse Transcriptase,Infectivity Assays, PCR based assays Adventitious Viruses: In Vitro & In Vivo Assays for viral contaminants Specific Virus Assays: Mouse Antibody (MAP) or Hamster Antibody (HAP) Production Tests; PCR assays for human or other viruses Other Virus Assays: Specific Bovine and Porcine virus assays

Working Cell Bank:

Normally, the WCB originates from a well characterised MCR, so only limited testing is required: Microbiological contaminants: Sterility, mycoplasma Cell Line Identification: Isoenzyme analysis or DNA fingerprinting or karyology Adventitious Viruses: In Vitro and In Vivo Assays for viral contaminants

Cells at the Limit of in vitro Cell Age or End of Production Cells

These cells should be evaluated once for those endogenous viruses which may not have been detected in the MCB and WCB. Also by conducting adventitious virus assays once, shows that the production process is not prone to such contamination. Microbiological contaminants: Sterility, mycoplasma Cell Line Identification: Isoenzyme analysis or DNA fingerprinting or karyology Retroviruses: Reverse Transcriptase, Infective Assays, PCR based assays Adventitious Viruses: In Vitro and In Vivo Assays for viral contaminants Other Virus Assays: Specific Bovine and Porcine viral assays

Unprocessed Bulk

A representative sample of the unprocessed bulk, removed from the bioreactor prior to further processing, represents one of the most suitable levels to detect adventitious viral contaminants with a high probability of detection. The testing that should be considered includes: Microbiological contaminants: Sterility, mycoplasma Adventitious Viruses: In Vitro and In Vivo Assays for viral contaminants Estimation of Retroviral Load: For rodent cell lines in particular, an electron microscopy assay should be performed to estimate to the level of retroviral-like particles

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