Search

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

The process capability and potential for various forms of chromatography to remove viruses have been discussed extensively in the literature, including the observed variability in performance of some unit operations such as Protein A and cation exchange (CEX). Some unit operations such as anion exchange (AEX) have shown robustness over a wide range of operating conditions. The robustness and effectiveness of the AEX step combined with a detailed understanding of the mechanisms that result in virus and impurity partitioning versus protein partitioning (e.g. conductivity, loading, pH range) support the feasibility of a generic or modular claim for AEX. A more fundamental understanding of the mechanisms for other chromatographic media (Protein A, CEX, and mixed-mode) could lead to more effective and more robust log reduction value (LRV) claims for these steps as well. Specific examples of CEX and mixed-mode chromatography were explored in the session and were also discussed in detail at the 2013 and 2015 Viral Clearance Symposia. Although some gaps remain in the mechanistic understanding of these unit operations, significant progress has been made and was reported at the 2017 Viral Clearance Symposium. It is important to note that recent publications on the mechanisms of viral clearance for mixed-mode chromatography and a framework for measurement of relative hydrophobicity have provided insights and new tools to better define the operating space and critical process parameters. The session also explored the use of next-generation mixed-mode adsorbers and the potential mechanisms contributing to the observed viral clearance. Gaps were also identified (e.g. integrity test when size-based mechanisms are used) and should be addressed to ensure robust viral clearance for these integrated and productive emerging unit operations. LAY ABSTRACT: Preparative chromatography is the most widely used unit operation for purification of therapeutic proteins. This session focused on the potential for various forms of chromatography to remove viruses. To advance to the next level of process, understanding the virus removal mechanism of different types of chromatography was addressed., (© PDA, Inc. 2018.)

The manufacturing processes of biological medicinal products are expected to be capable of removing and/or inactivating viruses, to primarily provide for adequate safety margins to patients and to thus satisfy the corresponding regulatory expectations. To achieve this goal, process segments specifically dedicated to the task of virus removal or inactivation are designed into manufacturing flows. The state of the art now includes virus removal by nanofiltration, and the more traditional low pH or (solvent-) detergent treatments are used widely, often to provide for two complementary and mechanically different means of virus clearance. Reflective of these process preferences, the virus-filtration/-inactivation session included seven case studies on virus filtration, and one each for a detergent and a low pH treatment. LAY ABSTRACT: To enhance virus clearance capacity, manufacturing processes of therapeutic proteins include process steps dedicated to virus clearance, especially virus-retentive filtration and virus inactivation. This article summarizes the current understanding of process preferences for the said process steps., (© PDA, Inc. 2018.)

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 removal by filtration has been an important improvement for the safety margins of plasma-derived medicinal products and has become a standard manufacturing process step for recombinant proteins. While the mechanism of action was initially considered to be strictly size-based, it has recently been recognized that a more complex interaction of the specific filter membrane and its pore architecture with filtrate flow rates may potentially influence the level of virus removal. Based on this improved understanding, parameters beyond the traditional state-of-the-art may need to be included into the design and control of these processes, and the validity of virus removal data generated in small-scale models for the manufacturing scale processes may need to be reevaluated. This article presents a tool for the analysis of flow rate during manufacturing or virus-spiked small-scale runs with a focus on the effects of low or no flow (stop) situations on the robustness of virus removal., Lay Abstract: Virus removal by filtration has improved safety margins of plasma-derived medicinal products and recombinant proteins. However, low or no flow (stop) situations during virus filtration may potentially affect virus removal. Transforming filtrate flow versus time data into histograms generates intuitively understandable visuals that provide a powerful tool to investigate the equivalence of manufacturing scale processes and down-scaled virus-spiked study runs, and thus to understand the robustness of virus removal for this parameter. With this tool at hand, lower mean flow rates during small-scale experiments are an elegant approach to support the robustness of virus filtration with respect to this parameter at manufacturing scale., (© PDA, Inc. 2016.)

The testing for adventitious viruses is of critical importance during development and production of biological products. The recent emergence and ongoing development of broad virus detection methods calls for an evaluation of whether these methods can appropriately be implemented into current adventitious agent testing procedures. To assess the suitability of several broad virus detection methods, a comparative experimental study was conducted: four virus preparations, which were spiked at two different concentrations each into two different cell culture media, were sent to four investigators in a blinded fashion for analysis with broad virus detection methods such as polymerase chain reaction-electrospray ionization mass spectrometry (PCR-ESI/MS), microarray, and two approaches utilizing massively parallel sequencing. The results that were reported by the investigators revealed that all methods were able to identify the majority of samples correctly (mean 83%), with a surprisingly narrow range among the methods, that is, between 72% (PCR-ESI/MS) and 95% (microarray). In addition to the correct results, a variety of unexpected assignments were reported for a minority of samples, again with little variation regarding the methods used (range 20-45%), while false negatives were reported for 0-25% of the samples. Regarding assay sensitivity, the viruses were detected by all methods included in this study at concentrations of about 4-5 log10 quantitative PCR copies/mL, and probably with higher sensitivity in some cases. In summary, the broad virus detection methods investigated were shown to be suitable even for detection of relatively low virus concentrations. However, there is also some potential for the production of false-positive as well as false-negative assignments, which indicates the requirement for further improvements before these methods can be considered for routine use., (© PDA, Inc. 2014.)

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)