Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus.

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10(10) PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4-5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3-101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79-85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic-Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V75). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less-pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log10 . A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V75 for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography).

Effectiveness of various processing steps for viral clearance of therapeutic proteins: database analyses of commonly used steps.

The successful implementation of any biologically derived product in human clinical trials and as a marketed biopharmaceutical requires the critical utilization of effective viral clearance steps. As biologic products have inherent risks of potentially carrying and or amplifying adventitious viruses that may be present in or introduced into the original materials, a number of processing steps are needed to provide adequate virus removal. Some common process steps are introduced into downstream purification schemes that provide a physical means to separate and/or remove viruses from the therapeutic protein. The physical steps often include virus-removing filters and chromatographic resins in column or membrane configurations, but can also include the introduction of irradiation, high heat steps, or other means for destroying the infectivity of a virus. Chemical treatment steps are often utilized as a means to inactivate a wide variety of virus types. A general overview is provided that describes the most commonly used techniques for virus removal or inactivation for the validation of virus clearance. Data sets from studies performed at WuXi AppTec for a wide variety of biologics reveal a number of steps that provide guidance for the design of process steps dedicated to viral clearance. The overall efficiency of several process steps reveals a number of efficient, robust steps, such as nanofiltration which can be designed for removal of almost all viral species. Exposure to a low pH or solvent detergent is also a robust step for inactivating enveloped virus. Steps with greater variances in predictability include chromatography steps such as capture columns and anion exchange resins. A lower removal capacity is typically expected for other chromatography steps such as cation exchange steps.