Effective inactivation of a wide range of viruses by pasteurization

Gröner, Albrecht; Broumis, Connie; Fang, Randel; Nowak, Thomas; Popp, Birgit; Schäfer, Wolfram; Roth, Nathan J.

The overall results demonstrate that, under the specific conditions optimized for and implemented in each manufacturing process, pasteurization is a highly robust and broadly effective virus inactivation step. Heat inactivation methods such as pasteurization and dry heat treatment thermally destabilize the intermolecular interactions between virus capsid proteins (and/or the integrity of the virus envelope where relevant) thereby resulting in a loss of virion capsid structural integrity and virus infectivity. 17 This contrasts to solvent/detergent or caprylate (octanoic acid) inactivation methods that destabilize or destroy the lipid envelope only, not the virus capsid, and are therefore effective for enveloped viruses only. 15 Pasteurization effectively (>4 log) inactivated all family members of enveloped viruses studied, evaluated under both target and robustness conditions. All enveloped viruses, except PRV, were inactivated to below the LOD at time points before the 10-hour production minimum, providing a margin of safety and indicating that the true virus inactivation capacity of the step was likely higher than represented by the log RFs that were demonstrated in the virus validation studies. PRV, a model for herpes viruses and other large DNA enveloped viruses, was also effectively inactivated by pasteurization but with slower inactivation kinetics and the presence of residual infectivity. The slower kinetic rates of PRV inactivation was directly related to sucrose concentration, which illustrates the balance between virus inactivation and protection of therapeutic protein activity; high sucrose concentrations were only employed in processes when required to preserve therapeutic protein functionality. Human herpes viruses studied were inactivated faster than PRV, verifying the use of PRV as a worse-case model virus in virus validation studies. It should be noted that PRV and herpesviruses are large and, therefore, effectively removed by small-(15-20 nm) and large-pore (35-75 nm) virus filtration, partitioned by fractionation steps, and are susceptible to other inactivation steps (e.g., Dichtelm€ uller et al. 18, 19 ) . Nonenveloped viruses are susceptible to inactivation by pasteurization. HAV was reliably and effectively inactivated by heat treatment in albumin and in stabilized product intermediates, in line with published data. 6, 16 However, the HAV strain HM 175, substrain 24a, is reported to have a higher heat sensitivity than other substrains, at least when pasteurized in albumin. 20, 21 Studies using the more heat-resistant HAV substrain 18f demonstrate no or little impact on inactivation kinetics for sucrose stabilized product intermediates (data not shown, manuscript in preparation). Only the nonenveloped animal parvoviruses, CPV and MVM, showed broad resistance to heat treatment (1-1.5 log), whereas a slightly higher virus RF of up to 3 log was achieved in the presence of immunoglobulins. Interaction between immunoglobulins and the virus may result in destabilization of the virus capsid, similar to that which is described for heat treatment of human parvoviruses. 22 In contrast, the human pathogenic parvovirus B19V is effectively inactivated in most product intermediates, consistent with previous data. 17, 22 An exception is the ATIII intermediate where high sucrose and CaCl 2 concentrations may be the factors that stabilize B19V; this is in line with Boschetti et al. 23 Virus reduction steps within biologic manufacturi