By Kathryn Martin Remington, Ph.D., Principal Scientist for Clearance Services, BioReliance
When evaluating viral reduction for a given process step, the reduction is the difference between the total virus in the input sample and the total virus in the postpurification, product-containing fraction. The results of viral assays are typically provided as a titer; that is viral units (e.g., TCID50, PFU, genome equivalents, etc.) per unit volume. For calculation of total virus in each fraction, the volume of each fraction be must be considered, and this is done by multiplying the volume of the input and output fractions by the virus titers. Virus reduction, then, can be calculated as (ICH Topic Q5A (R1) Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, CPMP/ICH/295/95).
The reduction for each purification step in the manufacturing process that is evaluated for viral clearance can be summed to determine the overall process reduction. Due to the inherent variability in the biological assays used for virus detection, however, reductions of one log or less cannot be included in the overall process reduction.
Virus reduction is expressed on a logarithmic scale, and while virus can be reduced to very low levels, it can never be reduced to zero. If no virus is detected in a sample, a theoretical endpoint is calculated. Using the Poisson distribution, the concentration of virus that would be present in the sample in order for the small volume that is tested in the virus assay to not contain a virus particle 95% of the time (Figure 1).