Is Clarence calculating clearance correctly?

by Dr. Ray Nims

As pointed out by Dr. Rudge in a recent posting “Do we have clearance, Clarence?”, spiking studies conducted for the purpose of validating impurity clearance are often done at only one spiking level (indeed often at the highest possible impurity load attainable). This is especially true for validation of adventitious agent (virus and mycoplasma) clearance in downstream processes. The studies are done in this way in order to determine the upper limit of agent clearance (in terms of log10 reduction) by the process. Such log10 reduction factors from individual process steps are then summed in order to determine the overall capability of the downstream processes to clear adventitious agents. The regulatory agencies have fairly clear expectations around such clearance capabilities which must generally be met by biologics manufacturers.

The limiting factor in such clearance studies is typically the amount or titer of the agent that is able to be spiked into the process solution, which is determined by: 1) the titer of the stock used for spiking, and 2) the maximum dilution of the process solution allowed during spiking (typically 10%). Under these circumstances, as Scott points out, there is a possibility that the determined clearance efficiency (i.e., the percentage of the load which is cleared during the step) is an underestimate of the actual clearance that might be obtained at lower impurity loading levels.

Adventitious agent clearance is comprised of two possible modalities, removal and inactivation. Removal refers to physical processes designed to eliminate the agent from the process solution, usually through filtration or chromatography. Removal efficiency through filtration would not be expected to display variability based on impurity loading. On the other hand, chromatographic separation of agents (by, for example, ion-exchange columns) may display saturation at the highest loadings, and therefore use of the highest possible loading levels may result in underestimates of removal efficiency at lower (i.e., more typical) impurity levels.

Inactivation refers to physical or chemical means of rendering the agent non-infectious. Agent inactivation is not always a simple, first-order reaction. It may be more complex, with a fast phase 1 stage of inactivation followed by a slow phase 2 stage of inactivation. An inactivation study is planned in such a way that samples are taken at different times so that an inactivation time curve can be constructed. As with removal studies, the highest possible impurity levels are typically used to determine inactivation kinetics.

Source: Omar et al. Transfusion 36:866-872, 1996

While the information obtained through clearance studies of this type may be incomplete from the point of view of understanding the relationships between impurity loading levels and clearance efficiency, the results obtained are consistent with the regulatory expectation that the clearance modalities be evaluated under worst-case conditions. Therefore, at least in the case of adventitious agent clearance validation, I would say that Clarence is calculating clearance correctly!