USP Viral Clearance and Test Methodologies
September 17, 2011
Virology test methods are increasingly important for the assessment of safety and effectiveness of medical treatments. For example, the development of biological (biologics and biotechnology-derived) products and therapies for human or animal use has created the need for sensitive viral detection assays for use in the GMP production and testing of biological products. This need is not limited to the production of viral vaccines, but also applies to the development and manufacture of recombinant proteins, cell and gene therapies, and other products.
The purpose of the virology test methods is for quality control testing of a variety of raw materials and processing reagents of animal origin used to manufacture biological products that have varying potential for introducing viral contaminants. According to USP <1237> VIROLOGY TEST METHODS,
“The production of biological products may allow the replication of adventitious agents during processing, and therefore these materials must be prescreened to avoid the opportunity for contamination of the product. Another point to consider regarding screening these materials is that the product may not be compatible with processing methods used to eliminate or inactivate these adventitious agents. Because of the nature of the biological products, the production process needs to include appropriate testing regimens that monitor the possible introduction of adventitious agents and/or viral agents into the systems used. For these reasons, sensitive viral detection methods are required not only for the release testing of biological drug products, but also during the intermediate stages of processing, process development, and routine manufacture. Important stages for consideration include the development of cell substrates and banks, raw materials of animal origin, process intermediates, and critical excipients when derived from animal tissues. This strategy should be augmented with viral clearance and inactivation studies whenever possible.”
There are several aspects to virology testing and all of them are complex requiring expert knowledge and sophisticated laboratories. In the purest sense one demonstrates the presence of a virus by growing it in a host such as cell culture, chicken embryo, or an animal [e.g., mouse, duck, rhesus monkey]. Infectious virus particles contaminating biologics and biotechnology-derived products are of great safety concern, because they have the potential for causing serious, possibly life-threatening, infections in the patients treated. This is particularly true if the patients are immunocompromised.
The purpose of a viral clearance study is to obtain assurance that a significant safety margin can be established through viral detection methods applied to unprocessed bulk and raw materials before purification in combination with purification processes that demonstrate the ability to inactivate or remove potential viral contaminants present at levels too low to detect. (See Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin <1050> for information on viral clearance and inactivation methods.)
Careful sample preparation is critical to validate viral clearance because the virus that one wants to demonstrate being cleared may be labile. Therefore, sample preparation typically involves storage of test samples at low temperatures (ideally -60 or below) as soon as practicable upon collection. When intended for use in a viral screening assay, aliquots of samples should be prepared to avoid multiple freezing and thawing. Samples intended for viral infectivity assays are typically shipped with sufficient dry ice to last several days more than the expected time required for transit. When received at the testing laboratory, the sample should be examined to verify that it is still frozen, and appropriate documentation should be completed. For any storage or hold condition, the impact of the condition on viral viability should be empirically assessed and sufficient cold chain management ensured.
Typical sample types for viral detection assays are:
- Cell Lysates— Test samples derived from cell substrates (master and working cell banks, end-of-production cell samples) are prepared in a manner that allows sampling of both the cells (for cell-associated viruses) and the conditioned medium (for virus shed into the medium). To achieve this, a culture of the cells is sampled. A cell suspension of ~107 cells per mL in conditioned medium is prepared and frozen (ideally at -60 or below). Because this medium does not contain cryopreservative, the majority of the cells will lyse upon thawing of the sample, releasing the cell-associated virus. Low-speed centrifugation will remove larger cellular debris and yield a supernatant that may be inoculated directly onto detector cells in cell-based viral infectivity assays.
- Biotechnology Bulk Harvest (Unprocessed Bulk Harvest) Samples— Routine lot testing of bulk harvest samples is mandatory for most types of biologics.
- Raw Materials of Animal Origin— Ingredients of animal origin used in the manufacture of biological products for human or veterinary use must be tested for species-specific viruses of concern as described in 9 CFR 113.53. The raw materials may be stored under a variety of conditions, as appropriate to the raw material. Sample preparation and method of application to the test system depend on the nature of the sample.
Gibraltar has considerable expert knowledge in the classical and molecular procedures necessary to achieve compliance. We have published many papers and maintains one of the private largest repositories of cells and viruses for the above purposes.
 “The Principles of Viral Control and Transmission”; Herbert N Prince, Daniel L. Prince, Richard N. Prince; Disinfection, Sterilization and Preservation, 4th Edition; 1991;
“Quality Assurance of Monoclonal Products: Virologic and Molecular Biologic Consideration”; Daniel L. Prince, Richard N. Prince; Journal of Industrial Microbiology, 3 (1988) 157-165; 1988 (MV); ” Methodological Approaches to Disinfection of Human Hepatitis B Virus”; D.L. Prince, H,N. Prince; O. Thraenhart, E. Muchmore, Ebonder, J. Pugh; Journal of Clinical Microbiology, P.3296-3304;Dec.93;
Prince et al. (2011) “Relationship of subtype influenza A pandemic strains to virucidal activity of a quaternary ammonium disinfectant.” Influenza and Other Respiratory Viruses 5 (Suppl. 1), 301-327.
Dr. Daniel Prince is president of Gibraltar Laboratories Inc. He has led the Company’s expansion and growth since 1987.
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