A major concern for many biologics is viral contamination. The ICH Q5A regulatory guideline indicates that a manufacturer of biological products for human use should demonstrate the capability of the manufacturing process to remove or inactivate known contaminants. One aspect of assessing the viral safety of a biopharmaceutical is to perform a viral clearance (or virus validation) study. The aim of a viral clearance study is to assess the effectiveness of individual steps in the manufacturing process at removing or inactivating viruses. These data are used to give a quantitative estimate of the overall level of virus clearance obtained by the manufacturing process. A similar approach can be used to demonstrate the removal of other potential contaminants, e.g. DNA, mycoplasma, endotoxins and bacteria. For viral clearance studies infectivity assays are the preferred method to determine the effectiveness of a process step in removing and/or inactivating infectious agents. Valuable additional data on the partitioning of viruses can be obtained by the use of a quantitative polymerase chain reaction (Q-PCR) assay. In some chromatography processes, for example, the buffers used may result in virus inactivation. Although the inactivation kinetics of the buffer may be studied as an independent step, it may not be possible to demonstrate how the virus partitions during the chromatography process using infectivity assays. In other words, any physical removal of virus due to the chromatography process itself could not be studied. However, with the introduction of Q-PCR assays, many chromatography processes may now also be studied, at least with respect to removal or partitioning of virus genomes. Such studies would be performed in parallel with infectivity studies. At BioReliance we have validated Q-PCR assays for the majority of commonly tested viruses.

For a viral clearance study of a product entering phase I/II clinical trials, a minimum of a single study of a single virus should be performed. For biologics derived from human blood products, the single virus chosen would be HIV. MLV would be the single virus chosen to study products derived from rodent cell lines at phase I/II. Increasingly, regulatory authorities have been asking for a second robust virus to be included in such early phase studies to show, at an early stage, the overall robustness of the process. We would, therefore, often incorporate a parvovirus model.

Where a product is due to enter phase III clinical trials a study should demonstrate the capacity of a process to reproducibly remove a range of viruses with different physico-chemical properties. Thus a study would typically be performed in duplicate, and include a panel of 4 (for recombinant proteins and monoclonal antibodies) or 5 (for human blood products) viruses.

Regulatory authorities are now also asking to see data on the robustness of a process, looking at extremes of conditions such as pH, temperature, and protein concentration. These data give an indication of the virus reduction capabilities of a manufacturing process under worst case conditions.

At BioReliance, we have taken an active role in collaborating with biotech and biopharma companies in the development of new and improved assays for viruses and TSEs. The TSE Western blot assay has been in routine use at BioReliance since 1997 and is becoming increasingly more important as the need for TSE safety assessments of b iologics increases. This assay specifically detects the disease-associated prion protein, PrPSc (or PrP-res). Levels of PrPSc have been shown to correlate closely with levels of infectivity. The Western blot assay provides a rapid means of making a preliminary evaluation of the effectiveness of steps in the removal of TSE agents, and can reduce the need for more expensive, lengthy bioassays.

Examples of process steps studied for viral clearance include:

• Heat Treatment
  • Pasteurisation
  • Lyophilisation/dry heat
• Solvent Detergent
• pH Treatment
  • Low pH (column elution)
  • High pH (sanitisation)
• Precipitation
  • Ethanol
  • Polyethylene Glycol
• Chromatography
  • Ion Exchange
  • Affinity
  • Hydrophobic Interaction
  • Reverse Phase
• Virus removal filtration

Examples of products for which clearance studies have been performed include:

• human and bovine blood products and coagulation factors
• equine and ovine blood products
• human and animal urine derived products
• tissue derived products
• monoclonal antibodies
• recombinant proteins
• medical devices (including collagen-derived products)
• enzymes