Using Sensitivity Analysis to Simplify the Virus Safety Factor Calculation in the Manufacture of Biopharmaceuticals.

Virus safety of biopharmaceuticals produced in cells of animal origin is governed by regulatory guidelines. It is ensured through raw material controls, cell substrate testing, and evaluation of the purification process for virus clearance capability. An additional control for cell lines that contain endogenous viruses is the virus safety factor (VSF) calculation, to demonstrate that the virus clearance exceeds the amount of potential endogenous virus in a dose of product. Product-specific input data (product titer, process yield, intended dose, purification process virus clearance capability, and the measured titer of endogenous virus produced by the cells) are typically used for the calculation. A wide range of relevant data was obtained from the production of monoclonal antibodies in Chinese Hamster Ovary (CHO) cells, and a sensitivity analysis was performed by using Monte Carlo simulations to determine which input data had the most significant impact on the range and distribution of the VSF. The sensitivity analysis suggested that the VSF calculation can be streamlined to include virus clearance capability, the endogenous virus titer, and dose while excluding product titer and process yield. Furthermore, the simulated VSF exceeded 4 log10 in 96% of the simulations, providing a high level of assurance of virus safety for antibodies produced in CHO cells and purified within specified operational parameters.

Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development.

Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.

Virus removal robustness of ion exchange chromatography.

Virus removal by ion exchange chromatography enhances the safety profile of therapeutic protein products. The robustness of virus removal depends on electrostatic binding between virus and oppositely charged chromatography media. However, model retrovirus Xenotropic Murine Leukemia Virus (XMuLV) binding remains robust even when virus and media are both positively charged. We investigated this counter-intuitive phenomenon using side-by-side comparison of virus-media binding behavior of XMuLV versus parvovirus, two viruses very different in size and structure but comparable in isoelectric point. When both viruses were negatively charged, XMuLV bound to positive anion exchange media with higher strength than parvovirus. When both viruses were positively charged, XMuLV remained tightly bound to positive media but parvovirus was dissociated. Likewise, XMuLV binding to media was much stronger than parvovirus under cation exchange conditions. These findings suggest that XMuLV binding could be enhanced by localized charge distribution not possessed by parvovirus, which is an important consideration for designing chromatography processes with robust virus removal capacity.