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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 log 10 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., (© PDA, Inc. 2023.)

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particle were all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines., (© 2022 Wiley Periodicals LLC.)

Continuous chromatography is increasingly being used across the biotechnology industry due to its economic advantages. For adoption in commercial manufacturing, also models for virus clearance studies must be available. It is demonstrated how for a virus clearance study for a multispecific antibody, the continuous protein A capture chromatography process, being run on multiple interconnected columns, can be mimicked with only a single column. With this mimicking small-scale model, resources and complexity can be minimized, when conducting virus clearance studies at a contract research organization (CRO) lab. Obtained log reduction values (LRV) for murine leukemia virus (xMuLV) and minute virus of mice (MVM) virus, used as model viruses, are comparable to batch protein A chromatography and results described by other groups. The feasibility of this mimicking small-scale model helps to further reduce barriers to adoption when implementing continuous chromatography., (© 2022 Wiley-VCH GmbH.)

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters., (© 2020 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)

Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters., (© 2019 Wiley Periodicals, Inc.)

The discussion on facility risk mitigation was included for the first time at the 2017 Viral Clearance Symposium. A few topics were discussed in this session, including sanitization/cleaning against viruses, viral segregation, as well as the definition of a "functionally closed" system.Virus inactivation by disinfectants is critical for the biotechnology industry. The efficacy can differ, depending on whether applied to surfaces, in solutions, or in gas phases, as well as the respective disinfectants (i.e., peracetic acid/hydrogen peroxide-based, hypochlorite-based, or glutaraldehyde-based).Most equipment used in the biotech industry can be cleaned or sanitized by alkaline solutions. Many of these methods were studied regarding their virus reduction potential and were defined considering alkaline concentration, time, and temperature.Virus clearance may be compromised if cross contamination or carryover happens from an early step with potentially a higher level of virus to a later step in the purification process (i.e., after virus removal or inactivation). Critical potential carryover (V cpc ) is defined as the volume of carryover that will significantly affect the overall virus clearance of a purification process. Based on the evaluation of critical potential carryover, mitigation actions can be introduced to avoid such carryover.Appropriate segregation within manufacturing facilities is required by regulators and utilized by manufacturers to ensure that the final product has appropriate safety margins. However, consensus around basic definitions and approaches related to facility segregation is lacking. To address this gap, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing have begun a project with the goal of developing a definition for a "functionally closed" manufacturing system. LAY ABSTRACT: The discussion on facility risk mitigation was included for the first time at the 2017 Viral Clearance Symposium. The topics discussed in this session included sanitization/cleaning against viruses, viral segregation, as well as the definition of a "functionally closed" system.Virus inactivation by disinfectants is critical for the biotechnology industry. The efficacy can differ, depending on whether applied to surfaces, in solutions, or in gas phases, as well as the respective disinfectants.Most equipment used in the biotech industry can be cleaned or sanitized by alkaline solutions. Many of these methods were studied regarding their virus reduction potential and were defined considering alkaline concentration, time, and temperature.Virus clearance may be compromised if cross contamination or carryover happens from an early step with potentially a higher level of virus to a later step in the purification process (i.e., after virus removal or inactivation).Regarding segregation within manufacturing facilities, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing have begun a project with the goal of developing a definition for a "functionally closed" manufacturing system. During this session, the current definition was discussed., (© PDA, Inc. 2018.)

Virus clearance by depth filtration has not been well-understood mechanistically due to lack of quantitative data on filter charge characteristics and absence of systematic studies. It is generally believed that both electrostatic interactions and sized based mechanical entrapment contribute to virus clearance by depth filtration. In order to establish whether the effectiveness of virus clearance correlates with the charge characteristics of a given depth filter, a counter-ion displacement technique was employed to determine the ionic capacity for several depth filters. Two depth filters (Millipore B1HC and X0HC) with significant differences in ionic capacities were selected and evaluated for their ability to eliminate viruses. The high ionic capacity X0HC filter showed complete porcine parvovirus (PPV) clearance (eliminating the spiked viruses to below the limit of detection) under low conductivity conditions (≤2.5 mS/cm), achieving a log10 reduction factor (LRF) of > 4.8. On the other hand, the low ionic capacity B1HC filter achieved only ∼2.1-3.0 LRF of PPV clearance under the same conditions. These results indicate that parvovirus clearance by these two depth filters are mainly achieved via electrostatic interactions between the filters and PPV. When much larger xenotropic murine leukemia virus (XMuLV) was used as the model virus, complete retrovirus clearance was obtained under all conditions evaluated for both depth filters, suggesting the involvement of mechanisms other than just electrostatic interactions in XMuLV clearance., (© 2014 American Institute of Chemical Engineers.)

Abstract: Virus filtration membranes are used to obtain virus clearance during the purification of biopharmaceutical products. These direct flow (also referred to as dead end or normal flow) filtration membranes are designed to reject virus particles and yield >98% product recovery for proteins less than 170kDa. Virus filtration feed streams generally have high purity and high product concentrations. Decrease in permeate flux during virus filtration, which reduces filter capacity, is most likely due to fouling by small aggregates of the product species present in the feed stream. Retrovirus and parvovirus filtration membranes, as well as two ultrafiltration membranes with NMWCOs of 300 and 10kDa (kgmol−1), were tested using feed streams consisting of minute virus of mice (MVM) in the presence and absence of 1% (w/v) BSA. A novelty of our work comes from the fact that we directly compare the performance of these membranes using realistic model feed streams consisting of MVM (an FDA recommended model virus) and BSA. This approach provides an industrially relevant benchmark of the engineering performance of virus filters with respect to a limited set of structural and operational variants. All asymmetric membranes were operated in direct flow, constant pressure mode with the filtration surface (i.e. skin surface) facing away from the feed stream as is industrial practice for virus filtration. In addition, the 10kDa ultrafiltration membrane was also operated with the skin surface facing the feed stream as is industrial practice for ultrafiltration. While little rejection of virus particles was observed for the 10kDa membrane when the skin surface faced away from the feed stream, significant (in excess of 3 log) virus rejection was observed when the skin surface faced the feed stream. Changes in permeate flux with filtrate volume were determined along with parvovirus rejection. Decreases in permeate flux were shown to result from fouling in the presence of BSA. No decrease in permeate flux was observed in the absence of BSA. The decrease in permeate flux has been analyzed in terms of classical pore blockage models. The results of this work, which compare the performance of virus filtration and similar ultrafiltration membranes, provide insights into designing virus filtration membranes. [Copyright &y& Elsevier]

Abstract: Viral clearance studies for naïve and maximally cycled chromatographic resins used for cGMP recombinant protein production are reviewed for three products, comprising 10 different chromatographic steps, including affinity, ion exchange, immobilized metal ion affinity, and hydrophobic interaction modes. Thirty-two separate studies were conducted (over 90 runs in total). No consistent reductions in model virus clearance were observed with used resins. The results address the reproducibility of virus clearance studies conducted by different scientists over several years at multiple contract labs. The log reduction values (LRVs) are typically within 0.5LRVs for new and used resin, but varied as much as 2LRVs for resins showing no functional deterioration. This relatively large difference is not believed to reflect resin changes, but highlights the challenges encountered in modeling column clearance. Production column performance and cleaning efficacy are demonstrated for these steps by trending mock runs, impurity removal and product recovery. No deterioration in cGMP column performance is seen over the established resin lifetimes, confirming that the resin regeneration and sanitization procedures restore the resins to a suitable initial state without damage. It is proposed that for some chromatography steps, the combination of lab-scale cycling studies confirming consistent performance throughout the resin lifetime and monitoring of cGMP manufacturing preclude the need for virus clearance studies on maximally cycled resin. [Copyright &y& Elsevier]