Virus filtration: A review of current and future practices in bioprocessing.

For drug products manufactured in mammalian cells, safety assurance practices are needed during production to assure that the final medicinal product is safe from the potential risk of viral contamination. Virus filters provide viral retention for a range of viruses through robust, largely size-based retention mechanism. Therefore, a virus filtration step is commonly utilized in a well-designed recombinant therapeutic protein purification process and is a key component in an overall strategy to minimize the risks of adventitious and endogenous viral particles during the manufacturing of biotechnology products. This study summarizes the history of virus filtration, currently available virus filters and prefilters, and virus filtration integrity test methods and study models. There is also discussion of current understanding and gaps with an eye toward future trends and emerging filtration technologies.

Multi-Attribute Method for Quality Control of Therapeutic Proteins.

Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

Validation and optimization of viral clearance in a downstream continuous chromatography setting.

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.

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures.

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.

Comparison of purification strategies for antibodies used in a broad spectrum host cell protein immunoassay.

Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in bulk preparations of biotechnologically produced medicines. Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the polyclonal antibodies (pAbs) used. These pAbs are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. The use of protein A or protein G affinity chromatography results in purified pAbs that are not entirely HCP-specific, while the use of HCP affinity chromatography results in a more specific pAb population but may be harder to recover fully. In theory, both approaches have advantages and disadvantages for generating optimal reagents. In this study, we compared reagents from these two purification procedures using the same starting material, as well as those from a step-wise combination of the two by evaluating purity, concentration, reagent coverage by Western blotting, and performance in an enzyme-linked immunosorbent assay (ELISA). This study demonstrates that pAbs purified by each of the methods are very similar in terms of sensitivity, the ability to recognize a broad range of HCPs, and overall performance in an ELISA measuring a range of HCPs in upstream process and final drug substance (DS) samples.

Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.

A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS10+ -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS10+ to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS10+ -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS10+ for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

Adapting viral safety assurance strategies to continuous processing of biological products.

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.

A step-wise approach to define binding mechanisms of surrogate viral particles to multi-modal anion exchange resin in a single solute system.

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.

Post-translational structural modifications of immunoglobulin G and their effect on biological activity.

The size, heterogeneity, and biological production process of protein therapeutics like monoclonal antibodies create unique challenges for their analysis and regulation compared with small molecules. Complete structural characterization of a molecule 1000-fold heavier than aspirin is no small feat. Biological post-translational modifications such as glycosylation further complicate their characterization and regulation. Even approved protein therapeutics are known to contain multiple structural variants in differing amounts. Structural modification occurs during production and storage as well as within patients after administration. Thus, the goals of manufacturers and regulators are to control and characterize this heterogeneity, not take on the impossible task of eliminating it. The aim of this review is to describe the structural heterogeneities known to occur with immunoglobulin G (IgG), note current detection and analytical strategies, establish their causes, and define their potential effects on the ultimate safety, purity, and potency of antibody therapeutics when known.

Quality by design approach for viral clearance by protein a chromatography.

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.

Evidence for grow-through penetration of 0.2-µm-pore-size filters by Serratia marcescens and Brevundimonas diminuta.

We find that both Brevundimonas diminuta and Serratia marcescens can grow through sterilizing grade filter membranes of different membrane polymer compositions. Although this passage does not occur on a consistent basis, generation of "grow-through positive" results indicate that grow-through can occur stochastically at basal levels. This observation argues that the following risk mitigation strategies during pharmaceutical aseptic processing are warranted: minimization of processing times, and monitoring, minimizing and characterizing pre-filter bioburden.

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools.

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k(L)a) of sensor enabled static and rocking T-flasks is presented and compared with other non-instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k(L)a) in rocking T-flasks. We also carried out a scale-down study at matched k(L) a between a rocking T75-flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale-down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T-flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T-flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k(L)a) and disposability, we argue that rocking T-flasks can be readily integrated with wave bioreactors, making the transition from research-scale to manufacturing-scale a seamless process.

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool.

In this study, we perform mass transfer characterization (k(L) a) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch-based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5-fold resulted in 30% increase in k(L) a (from 2.3 to 3.0 h(-1) ) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control.

Physical characterization of icosahedral virus ultra structure, stability, and integrity using electrospray differential mobility analysis.

We present a rapid and quantitative method to physically characterize the structure and stability of viruses. Electrospray differential mobility analysis (ES-DMA) is used to determine the size of capsomers (i.e., hexons) and complete capsids. We demonstrate how to convert the measured mobility size into the icosahedral dimensions of a virus, which for PR772 become 68.4 nm for vertex-to-vertex, 54.4 nm for facet-to-facet, and 58.2 nm for edge-to-edge lengths, in reasonable agreement with dimensions from transmission electron microscopy for other members of the family Tectiviridae (e.g., PRD1). These results indicate ES-DMA's mobility diameter most closely approximates the edge-to-edge length. Using PR772's edge length (36.0 nm) and the size of the major capsid hexon (≈8.4 nm) from ES-DMA with icosahedral geometry, PR772's T = 25 symmetry is confirmed and the number of proteins in the capsid shell is determined. We also demonstrate the use of ES-DMA to monitor the temporal disintegration of PR772, the thermal degradation of PP7, and the appearance of degradation products, essential to viral stability assays. These results lay groundwork essential for the use of ES-DMA for a variety of applications including monitoring of vaccine and gene therapy vector products, confirmation of viral inactivation, and theoretical studies of self-assembling macromolecular structures.

Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.

Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The "gold standard" microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely 'go/no-go' decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates.

Industry and regulatory experience of the glycosylation of monoclonal antibodies.

We surveyed 23 antibody-related marketing applications for glycoform analytical and functional information. Our database analysis shows a clear trend of increasing sophistication of analytical methods used to identify and quantify glycans. These have revealed a high degree of complexity and heterogeneity of glycans attached to antibody products. The nature of the complexity is influenced by product type and expression system, and may be associated with functional consequences in some but not all cases.

Analysis of viral clearance unit operations for monoclonal antibodies.

Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody-related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late-phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro- and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log(10) were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses.

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask.

Routine cell culture is done in small-scale disposable vessels (typically 0.1-100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered "black boxes" because they are generally not equipped with sensors. In this study, we show that on-line monitoring of dissolved oxygen (DO) and pH in a T-75 flask-based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on-line monitoring of DO and pH show three distinctive metabolic regions in passages 1-18, 19-28, 29-54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (q(O2)), and the lactate production rate to the oxygen consumption rate yield (Y(Lac/ox)) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre- and seed culture prior to scale-up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques.

High Performance Size Exclusion Chromatography and High-Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products.

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10-12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1-1000 nm) in the submicron size range.