Early planning is the key to a smooth process
By Merribeth Morin, M.D., Director of Virology at Microtest Labs
Many drugs in clinical trials and on the market are characterized as biologicals. Biological drugs are those which are derived from living organisms, such as bacteria, yeast, mammalian cells, animals, and humans. The regulatory agencies have provided guidance documents and regulations that describe the requirements for virus testing of biological products. A thorough testing strategy is key to gaining regulatory approval.
The planning for the virus testing should begin early in the development of a biological product. This will help in assessing the safety of the product and in determining the complete panel of testing that will be needed. It is important to have solid documentation of the source and history of the materials involved. If there is any missing information, it is better to be aware of it early in the development process so that any required changes would have minimal impact.
Sources of viral contaminants
The manufacture of a biological product is often a very complex process. For many biological products, cell lines are engineered to express a particular protein or possess a certain trait. These cell lines are expanded and often banked in a tiered system producing both master and working cells banks. Cells from the working cell bank are expanded for use in a bioreactor to manufacture the product on a large scale. The bulk material from the bioreactor is then often used in a purification process to isolate the final drug product. For these types of biological products, one can see many opportunities for virus contamination, including: 1) introduction of viruses from the raw materials used in the culture of the cells, 2) source cells infected without any outward signs of contamination, 3) low-level virus not detected in the raw material screen or the tests of the cell banks amplified during the manufacturing process, 4) accidental operator introduction of viruses into the system during the manufacture and the processing steps to prepare the final biological product.
Animal-derived raw materials
Often in the culture of mammalian cells, bovine serum and/or products derived from bovine sources are used in the culture medium. Other reagents used in cells often are animal derived as well. For example, typsin, used for passaging adherent cells, is often derived from a porcine source. The Code of Federal Regulations, Animals and Animal Products, Title 9 (9CFR)1, provides regulations for the testing of animal products and these are often followed to determine the safety of these materials for producing human drugs. The CFR is updated annually, though there have not been major changes in the requirements for serum products or porcine trypsin over the past few years.
European guidances from the EMEA, for both human and veterinary applications describe additional requirements for the testing of bovine serum, lending particular concern to potential contamination by pestiviruses, (specifically Bovine viral diarrhea virus [BVDV]).2,3 The guidance for human biologics was adopted and came in to operation in 2003.2 The guidance for veterinary products was first adopted in 2001, and has since been revised, with the latest revision coming into effect in 2006.3 The testing described in these documents must be considered if the intent is to market a biological product in Europe.
There has been an industry trend producing cell banks and biological products in serum-free conditions. The need for animal virus testing, however, is not necessarily precluded for these products. The possibility exists that the cells, though banked in serum-free conditions, have a history where animal products were used. Further, while the culture medium is serum-free, growth factors are sometimes added to the media to promote the propagation of the cells. Often, these growth factors are derived from bovine sources. The presence of these agents in the media will therefore make tests for animal viruses important for both the raw materials and the cultured cells.
The cells used in the production of biologics can be a source of contamination in the manufacturing process. Virus infection is not always apparent in cells. Some viruses are endogenous to cells, such as that found in murine cells. (Murine cells can contain endogenous retrovirus sequences and infectious retrovirus can be recovered from these cells.) Cells may also harbor a latent virus infection that only under certain conditions could express itself, such as that seen in herpes virus infections. Further, there can be an inapparent virus infection in the cells with no observable signs indicating a virus infection in the culture. Cells used for the production of a biologic must therefore be tested for the presence of adventitious agents.
Regulatory documents for the testing of cell lines include the Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals,4 from the FDA, the Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use,5 also from the FDA, and the Guidance on Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin,6 from the International Conference on Harmonisation (ICH). The documents cited above are from 1993, 1997, and 1998, respectively, but they are still cited in newer documents as providing the concepts that need to be addressed for virus safety. The documents are written as guidances, and do not provide specific methods on how to perform the tests. It will be the responsibility of the manufacturer to assure that the testing described in the documents is satisfied, and that current methods are utilized and current virus concerns addressed.
Virus testing of the bulk product is also required as amplification of viruses not detected in the screening of the raw materials or the testing of the cell banks could occur during the manufacturing process. Further, introduction of viruses via contaminated raw materials, or accidental exposure due to a breach in the process could occur further necessitating virus testing. Many of the same considerations for the cell bank testing apply for testing the bulk harvest.
Due to the risk of virus contamination, the manufacturing process for many therapeutic products often contains steps that can clear potential virus contaminants. The process steps utilized for clearing viruses include those for inactivation and for removal. If such steps are incorporated for preparing the final product, these steps must be validated for virus removal. Regulatory guidances provide information on for designing the validation studies.4,5,6
Considerations for novel biologic products
While therapeutic proteins prepared in cell lines is common for biologic drugs, there are other types of biologic drugs in development and commercial use that have different processes for production. These include therapeutic drugs made from animals, and vaccines. Some of the novel animal products used for treating human disease are prepared from transgenic animals. A guidance was written by the FDA addressing the concerns for these types of products (Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans).8 A more recent draft guidance was published by the FDA that addresses some of the safety testing required for vaccines (Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases).7 Some of the concepts indicated in the earlier guidances for biological products are reiterated in these two documents but these address some of the more specific issues for these types of products.
Types of virus tests
Many virus tests are cell-based assays where cells, susceptible to virus infection, are inoculated with the test sample to determine if any virus is present. These tests can be general tests where the inoculated cells are observed for cytopathic effect due to virus infection. The types of cells chosen for the test depend on the types of viruses that might be present in the sample. Determining which cells to use is part of the overall testing strategy for the product. Immunofluorescent antibody tests can be incorporated in cell-based assays to detect specific viruses, when required. Other types of cell-based tests for viruses may also be needed depending on the product on the types of potential virus contaminants.
Besides cell-based tests, additional types of virus tests are also required to characterize cell lines and test biological products. In vivo studies looking for evidence of virus infection, electron microscopy studies of cells and of materials harvested from the bioreactor, and molecular-based assays to detect specific viruses will also need to be incorporated into the overall testing strategy for the product.
Contracting out the testing
Due to the extent of the testing that is required to assure the safety of biological products, some manufacturers will need to outsource much of this work. Further, the regulatory framework for biological product testing can be complex. Contract testing companies are experienced in performing the required testing and can assist in determining a testing strategy for a biological product. As not all products are alike, when selecting contract testing suppliers, it is important to review their capabilities and how they can help with a specific product. While there are a general strategies for therapeutic products made in a particular cell line; this strategy might not be the appropriate testing strategy for all therapeutic products made in a similar cell line.
The testing of biological products is required to assure the safety of drugs used for treating human disease and there are many regulatory documents that provide a framework for the virus testing strategy. Planning the testing early can help in determining a testing strategy ensuring product safety and possibly streamline the process. From a virus contamination perspective, coupling a thorough testing of the source materials with a solid viral clearance in the final purification steps will help ensure the final drug product is safe for human use.
About the author: Dr. Merribeth Morin is the Director of Virology for Microtest Laboratories. She holds a PhD in Virology from Harvard University and has spent a number of years in research and contract testing. Her research included developing DNA vaccines for the treatment of influenza virus infection and other infectious diseases, and the development of an attenuated vaccine for Herpes simplex virus infection. She has over seven years of experience in virology and biosafety testing.
1Code of Federal Regulations 9 (9CFR), Animal and Animal products. (current year). Part 113.53. Requirements for ingredients of animal origin used for production of biologics.2Note for Guidance on the Use of Bovine Serum in the Manufacture of Human Biological Medicinal Products. CPMP/BWP/1793/02.3Requirements and Controls Applied to Bovine Serum used in the Production of Immunological Veterinary Medicinal Products. EMEA/CVMP/743/00-Rev.2.4Draft Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals. FDA CBER, 7/12/19935Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use. FDA CBER, 2/28/19976International Conference on Harmonisation (ICH) Guidance, Q5A: Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin (63 FR 51074; 9/24/1998)7Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans. FDA CBER 4/3/20038Draft Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases. FDA CBER 9/28/2006