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Continuous bioprocessing holds the potential to improve product consistency, accelerate productivity, and lower cost of production. However, switching a bioprocess from traditional batch to continuous mode requires surmounting business and regulatory challenges. A key regulatory requirement for all biopharmaceuticals is virus safety, which is assured through a combination of testing and virus clearance through purification unit operations. For continuous processing, unit operations such as capture chromatography have aspects that could be impacted by a change to continuous multicolumn operation, for example, do they clear viruses as well as a traditional batch single column. In this study we evaluate how modifying chromatographic parameters including the linear velocity and resin capacity utilization could impact virus clearance in the context of moving from a single column to multicolumn operation. A Design of Experiment (DoE) approach was taken with two model monoclonal antibodies (mAbs) and two bacteriophages used as mammalian virus surrogates. The DoE enabled the identification of best and worst‐case scenario for virus clearance overall. Using these best and worst‐case conditions, virus clearance was tested in single column and multicolumn modes and found to be similar as measured by Log Reduction Values (LRV). The parameters identified as impactful for viral clearance in single column mode were predictive of multicolumn modes. Thus, these results support the hypothesis that the viral clearance capabilities of a multicolumn continuous Protein A system may be evaluated using an appropriately scaled‐down single mode column and equipment. [ABSTRACT FROM AUTHOR]

A multi‐tiered approach to determine the binding mechanism of viral clearance utilizing a multi‐modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X‐MuLV, MVM, REO3, and PrV). First, virus spiked buffer‐only experiments were conducted to evaluate the virus's affinity for single mode and multi‐modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups. This mechanistic view agreed with the general surface characteristics of the different virus species in terms of isoelectric point and relative hydrophobicity values. This hypothesized mechanistic binding was then tested with commercially relevant, in‐process materials, in which competitive binding occurred between the load components (e.g., viruses, target product, and impurities) and the resin. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1019–1026, 2018 [ABSTRACT FROM AUTHOR]

ABSTRACT Multi-modal anion exchange resins combine properties of both anion exchange and hydrophobic interaction chromatography for commercial protein polishing and may provide some viral clearance as well. From a regulatory viral clearance claim standpoint, it is unclear if multi-modal resins are truly orthogonal to either single-mode anion exchange or hydrophobic interaction columns. To answer this, a strategy of solute surface assays and High Throughput Screening of resin in concert with a scale-down model of large scale chromatography purification was employed to determine the predominant binding mechanisms of a panel of bacteriophage (i.e., PR772, PP7, and ϕX174) to multi-modal and single mode resins under various buffer conditions. The buffer conditions were restricted to buffer environments suggested by the manufacturer for the multi-modal resin. Each phage was examined for estimated net charge expression and relative hydrophobicity using chromatographic based methods. Overall, PP7 and PR772 bound to the multimodal resin via both anionic and hydrophobic moieties, while ϕX174 bound predominantly by the anionic moiety. Biotechnol. Bioeng. 2017;114: 1487-1494. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT There has been a recent drive in commercial large-scale production of biotechnology products to convert current batch mode processing to continuous processing manufacturing. There have been reports of model systems capable of adapting and linking upstream and downstream technologies into a continuous manufacturing pipeline. However, in many of these proposed continuous processing model systems, viral safety has not been comprehensively addressed. Viral safety and detection is a highly important and often expensive regulatory requirement for any new biological product. To ensure success in the adaption of continuous processing to large-scale production, there is a need to consider the development of approaches that allow for seamless incorporation of viral testing and clearance/inactivation methods. In this review, we outline potential strategies to apply current viral testing and clearance/inactivation technologies to continuous processing, as well as modifications of existing unit operations to ensure the successful integration of viral clearance into the continuous processing of biological products. Biotechnol. Bioeng. 2017;114: 21-32. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Virus retentive filters are a key product safety measure for biopharmaceuticals. A simplistic perception is that they function solely based on a size-based particle removal mechanism of mechanical sieving and retention of particles based on their hydrodynamic size. Recent observations have revealed a more nuanced picture, indicating that changes in viral particle retention can result from process pressure and/or flow interruptions. In this study, a mechanistic investigation was performed to help identify a potential mechanism leading to the reported reduced particle retention in small virus filters. Permeate flow rate or permeate driving force were varied and analyzed for their impact on particle retention in three commercially available small virus retentive filters. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:959-970, 2016 [ABSTRACT FROM AUTHOR]

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750-757, 2015 [ABSTRACT FROM AUTHOR]

Separation of viruses and other contaminants from protein therapeutics using anion exchange membrane adsorbers is a successful new approach to viral clearance; however, the fundamental phenomena that control performance are not well understood. For example, the kinetics of adsorption to the anion exchange surface may limit clearance, but has yet to be characterized experimentally and mathematically. In the present study, surface plasmon resonance was used to determine the adsorption kinetics for five large biological molecules: phage PP7, phage ΦX174, phage PR772, thyroglobulin, and DNA. Rate constants were incorporated into a kinetic model of chromatography to illustrate the impact on performance. [ABSTRACT FROM AUTHOR]

Bacteriophage binding mechanisms to multi-modal anion exchange resin may include both anion exchange and hydrophobic interactions, or the mechanism can be dominated by a single moiety. However, previous studies have reported binding mechanisms defined for simple solutions containing only buffer and a surrogate viral spike (i.e. bacteriophage ΦX174, PR772, and PP7). We employed phage spiked in-process monoclonal antibody (mAb) pools to model binding under bioprocessing conditions. These experiments allow the individual contributions of the mAb, in-process impurities, and buffer composition on mechanistic removal of phages to be studied. PP7 and PR772 use synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group to bind multi-modal resin. ΦX174′s binding mechanism remains inconclusive due to operating conditions. [ABSTRACT FROM AUTHOR]

Abstract: A simple model based on changes in filter resistance and active area of the membrane has been used to model viral filtration. Viral particles have been modeled as colloidal particles disregarding any specific interaction and considering only passive transport in the system. The model is based on the assumption that purely steric interactions determine the ratio of concentration of viral particles inside the pore to concentration in solution at the pore mouth. Viral particles rejected by the membrane form a layer of high concentration near the membrane and this layer offers additional resistance to filtration. The membrane flux has been calculated by applying Darcy׳s law. The overall model involves use of six unknown parameters to account for cake formation, nature of virus, interaction between the virus and the membrane, and pore size. The breakthrough of the model virus, bacteriophage ϕX-174, through normal-flow virus filters using commercial process fluids has been chosen as the system used for model validation. The model has been fitted to the time profile of flux and the log reduction value (LRV) of viral particles across the different types of commercially available filters. The model will be useful when performing studies using scale down models for correlating LRV to flux decline. The model also provides us insights into the underlying mechanisms behind viral clearance achieved from the various commercially available viral filters. [Copyright &y& Elsevier]

Abstract: We evaluated viral clearance in multiply-cycled anion-exchange media run in flow-through mode. We found that anion-exchange columns do not lose viral clearance capacity after extensive re-use, if they are cleaned with recommended buffers that do not chemically degrade the media. In contrast, anion-exchange (AEX) columns that are not cleaned or are cleaned with buffers that chemically degrade the media lost viral clearance capacity after extended use. In these cases, other performance attributes that changed at the same time were increased band spreading, decreased DNA clearance and accumulating backpressure that prevented re-use past 80–120 cycles. Thus, our data suggests that flow through mode anion-exchange columns that are cleaned with recommended cleaning buffers, and periodically monitored for band spreading, DNA clearance and/or backpressure need not be re-evaluated for viral clearance at the end of the validated media lifetime. [Copyright &y& Elsevier]