This article appears in a special supplement, A Renaissance in Biomanufacturing:The Art of Purification, published in August 2013.
Current regulatory guidelines require at least two orthogonal steps for the inactivation and/or removal of viruses, thus different principles of separation/inactivation must be used in each method (2,3). Because viruses vary in size, charge, and the presence or absence of an envelope, the available methods differ in their effectiveness against particular types of virus; these factors must be integrated into the design space during the development of a process ahead of Phase I clinical trials. Manufacturers are also expected to deliver a virus clearance strategy that has been optimized for each product because biopharmaceuticals are often large, complex proteins that resemble smaller viruses in their physical and chemical properties. Virus clearance strategies must, therefore, be tailored to avoid product loss. The virus removal steps must then be tested against at least two model viruses representing those most likely to be present in the process stream (2-3) and should include an endogenous virus if relevant to the process. Ideally, an adventitious virus such as minute virus of mice (MVM) or porcine parvovirus (PPV) should also be tested, because these are the gold standards for size-dependent clearance steps using 20-nm filters (2-4). Removal/inactivation is usually demonstrated in spiking studies, where specific viruses are added deliberately to the process stream ahead of the relevant unit operations. Before Phase III clinical trials can be authorized, two further viruses must be tested if specific contaminants are likely in the process stream. The two testing schemes have different aims: general virus clearance is designed to test process robustness, whereas specific virus clearance using anticipated contaminants aims to ensure product safety (4).Although virus clearance should be built into the design space on a product-by-product basis, several robust and effective strategies have become established in the industry (5-6). Appropriate methods for virus inactivation include heating/pasteurization or solvent/detergent treatments (7), although these may also have a significant negative impact on some recombinant proteins and are only effective against enveloped viruses. More often, a low-pH hold (8) is used for enveloped viruses if this is compatible with the buffer conditions in the process (e.g., during the production of monoclonal antibodies); exposure to ultraviolet light in the UVC range is used to inactivate all viruses by cross-linking the nucleic acids at 254 nm (9). Virus removal methods physically separate virus particles from the feed stream, and the most suitable methods are chromatography, where virus particles are captured by adsorption (10), and retentive filtration using 20-nm filters, which eliminates even the smallest viruses by size exclusion (2,4). However, there are currently no common standards for virus filtration. Instead, it is left to manufacturers to show, on a case-by-case basis, that their virus clearance steps are acceptable and efficient.