Monoclonal antibody capture and viral clearance by cation exchange chromatography

Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step.     

Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production

The quality‐by‐design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design‐of‐experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus‐like particles from CHO‐cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

Viral clearance using disposable systems in monoclonal antibody commercial downstream processing

Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed.     

Porcine circovirus (PCV) removal by Q sepharose fast flow chromatography

The recently discovered contamination of oral rotavirus vaccines led to exposure of millions of infants to porcine circovirus (PCV). PCV was not detected by conventional virus screening tests. Regulatory agencies expect exclusion of adventitious viruses from biological products. Therefore, methods for inactivation/removal of viruses have to be implemented as an additional safety barrier whenever feasible. However, inactivation or removal of PCV is difficult. PCV is highly resistant to widely used physicochemical inactivation procedures. Circoviruses such as PCV are the smallest viruses known and are not expected to be effectively removed by currently‐used virus filters due to the small size of the circovirus particles. Anion exchange chromatography such as Q Sepharose^® Fast Flow (QSFF) has been shown to effectively remove a range of viruses including parvoviruses. In this study, we investigated PCV1 removal by virus filtration and by QSFF chromatography. As expected, PCV1 could not be effectively removed by virus filtration. However, PCV1 could be effectively removed by QSFF as used during the purification of monoclonal antibodies (mAbs) and a log₁₀ reduction value (LRV) of 4.12 was obtained. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1464–1471, 2013     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance During the Purification Of Mammalian Cell Substrate Derived Biotherapeutics

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.     

Clearance of porcine circovirus and porcine parvovirus from porcine-derived pepsin by low pH inactivation and cation exchange chromatography

The contamination of oral rotavirus vaccines by porcine circovirus (PCV) raised questions about potential PCV contamination of other biological products when porcine trypsin or pepsin is used in production process. Several methods can be potentially implemented as a safety barrier when animal derived trypsin or pepsin is used. Removal of PCV is difficult by the commonly used viral filters with the pore size cutoff of approximately 20 nm because of the smaller size of PCV particles that are around 17 nm. It was speculated that operating the chromatography step at a pH higher than pepsin's low pI, but lower than pIs, of most viruses would allow the pepsin to flow through the resin and be recovered from the flow through pool whilst the viruses would be retained on the resin. In this study, we investigated low pH inactivation of viruses including PCV Type 1 (PCV1) and PCV1 removal by cation exchange chromatography (CEX) in the presence of pepsin. Both parvovirus and PCV1 could be effectively inactivated by low pH and PCV1 could be removed by POROS 50HS CEX. The POROS 50HS method presented in this article is helpful for designing other CEX methods for the same purpose and not much difference would be expected for similar product intermediates and same process parameters. While the effectiveness needs to be confirmed for specific applications, the results demonstrate that both low pH (pH 1.7) and CEX methods were successful in eliminating PCV1 and thus either can be considered as an effective virus barrier.     

Molecular determinants of self-association and rearrangement of a trimeric intermediate during the assembly of a parvovirus capsid

The minute virus of mice (MVM) provides a simple model for the dissection of the molecular determinants of the self-assembly, stability, and dynamics of a biological supramolecular complex. MVM assembly involves the trimerization of capsid subunits in the cytoplasm; trimers are transported to the nucleus, where they suffer a conformational change and are made competent for capsid formation. Our previous study revealed that capsid assembly from trimers is dependent on stronger intertrimer interactions that are equally spaced in an equatorial belt surrounding each trimer. We have now targeted the interfaces between monomers within each trimer to identify the molecular determinants of trimerization and the rearrangement needed for capsid assembly. Twenty-eight amino acid residues per monomer were individually mutated to alanine to remove most of the stronger intersubunit interactions. The effects on trimer and capsid assembly and virus infectivity in cells were analyzed. No side chain was individually required for trimer assembly in the cytoplasm; in contrast, half of them were required to make the trimers competent for nuclear capsid assembly, even though none was close to intertrimer interfaces. These critical side chains are conserved and participate in extensive hydrophobic contacts, buried hydrogen bonds, or salt bridges between subunits. This study on MVM capsid assembly reveals that: (i) trimerization is a robust process, insensitive to removal of individual intersubunit interactions; and (ii) the rearrangement of the trimer intermediate required for capsid assembly is a global process that depends on the establishment of many interactions along the protein-protein interfaces within each trimer.     

Particle-based analysis elucidates the real retention capacities of virus filters and enables optimal virus clearance study design with evaluation systems of diverse virological characteristics

In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number-based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co-spiking of empty and genome-containing particles resulted in a decrease in the virus removal performance proportional to the co-spike ratio. This suggests that empty particles are captured in the same way as genome-containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log₁₀ difference in particle-to-infectivity ratios, there was a 20-fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity.     

Characterizing and enhancing virus removal by protein A chromatography

Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal.     

The prevention of an anomalous chromatographic behavior and the resulting successful removal of viruses from monoclonal antibody with an asymmetric charge distribution by using a membrane adsorber in highly efficient,anion-exchange chromatography in flow-through mode

Anion exchange (AEX) chromatography in the flow-through mode is a widely employed purification process for removal of process/product-related impurities and exogenous/endogenous viruses from monoclonal antibodies (mAbs). The pH of the mobile phase for AEX chromatography is typically set at half a unit below the isoelectric point (pI) of each mAb (i.e., pI − 0.5) or lower and, in combination with a low ionic strength, these conditions are usually satisfactory for both the recovery of the mAb and removal of impurities. However, we have recently encountered a tight binding of mAb1 to AEX resins under these standard chromatographic conditions. This anomalous adsorption behavior appears to be an effect of the asymmetric charge distribution on the surface of the mAb1. We found that mAb1 did not bind to the AEX resins if the mobile phase has a much lower pH and higher ionic strength, but those conditions would not allow adequate virus removal. We predicted that the use of membrane adsorbers might provide effective mAb1 purification, since the supporting matrix has a network structure that would be less susceptible to interactions with the asymmetric charge distribution on the protein surface. We tested the Natriflo HD-Q AEX membrane adsorber under standard chromatographic conditions and found that mAb1 flowed through the membrane adsorber, resulting in successful separation from murine leukemia virus. This AEX membrane adsorber is expected to be useful for process development because mAbs can be purified under similar standard chromatographic conditions regardless of their charge distributions.     

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[¹⁴C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.Abbreviations DEAE diethylaminoethyl - OMT O-methyltransferase - SAM S-adenosyl-L-methionine - TMV tobacco mosaic virus     

The origin of multiple polypeptides of molecular weight below 110 000 encoded by tobacco mosaic virus RNA in the messenger-dependent rabbit reticulocyte lysate

Multiple polypeptides encoded by tobacco mosaic virus (TMV) RNA in the messenger-dependent rabbit reticulocyte lysate are not attributable to contaminating 3′-coterminal RNA fragments, multiple leaky termination codons or endonuclease activity opening-up legitimate or spurious internal initiation sites. Quantitative analysis of polypeptides encoded over a range of added RNA concentrations from 0.09 μg·ml^?1 to 180 μg·ml^?1 compared wi preparation, or with RNA extracted from the alkali-stable fraction of TMV suggest that apart from four legitimate virus-coded products of apparent M_r approx. 165 000, 110 000, 30 000 and 17 500 all other polypeptides arise from the overlapping 5′-proximal cistrons either by (i) site-selective endonucleolytic cleavage, (ii) sense codon misreading, or (iii) specific regions of secondary structure on TMV RNA which impede ribosome translocation.     

The simplex algorithm for the rapid identification of operating conditions during early bioprocess development: case studies in FAb' precipitation and multimodal chromatography

This study describes a data-driven algorithm as a rapid alternative to conventional Design of Experiments (DoE) approaches for identifying feasible operating conditions during early bioprocess development. In general, DoE methods involve fitting regression models to experimental data, but if model fitness is inadequate then further experimentation is required to gain more confidence in the location of an optimum. This can be undesirable during very early process development when feedstock is in limited supply and especially if a significant percentage of the tested conditions are ultimately found to be sub-optimal. An alternative approach involves focusing solely upon the feasible regions by using the knowledge gained from each condition to direct the choice of subsequent test locations that lead towards an optimum. To illustrate the principle, this study describes the application of the Simplex algorithm which uses accumulated knowledge from previous test points to direct the choice of successive conditions towards better regions. The method is illustrated by two case studies; a two variable precipitation example investigating how salt concentration and pH affect FAb' recovery from E. coli homogenate and a three-variable chromatography example identifying the optimal pH and concentrations of two salts in an elution buffer used to recover ovine antibody bound to a multimodal cation exchange matrix. Two-level and face-centered central composite regression models were constructed for each study and statistical analysis showed that they provided a poor fit to the data, necessitating additional experimentation to confirm the robust regions of the search space. By comparison, the Simplex algorithm identified a good operating point using 50% and 70% fewer conditions for the precipitation and chromatography studies, respectively. Hence, data-driven approaches have significant potential for early process development when material supply is at a premium.     

Chromatography process development in the quality by design paradigm I: Establishing a high‐throughput process development platform as a tool for estimating “characterization space” for an ion exchange chromatography step

This article describes the development of a high‐throughput process development (HTPD) platform for developing chromatography steps. An assessment of the platform as a tool for establishing the “characterization space” for an ion exchange chromatography step has been performed by using design of experiments. Case studies involving use of a biotech therapeutic, granulocyte colony‐stimulating factor have been used to demonstrate the performance of the platform. We discuss the various challenges that arise when working at such small volumes along with the solutions that we propose to alleviate these challenges to make the HTPD data suitable for empirical modeling. Further, we have also validated the scalability of this platform by comparing the results from the HTPD platform (2 and 6 μL resin volumes) against those obtained at the traditional laboratory scale (resin volume, 0.5 mL). We find that after integration of the proposed correction factors, the HTPD platform is capable of performing the process optimization studies at 170‐fold higher productivity. The platform is capable of providing semi‐quantitative assessment of the effects of the various input parameters under consideration. We think that platform such as the one presented is an excellent tool for examining the “characterization space” and reducing the extensive experimentation at the traditional lab scale that is otherwise required for establishing the “design space.” Thus, this platform will specifically aid in successful implementation of quality by design in biotech process development. This is especially significant in view of the constraints with respect to time and resources that the biopharma industry faces today. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 403–414, 2013     

浙江武义2009年南方水稻黑条矮缩病的毒源地分析

2009年, 浙江省境内首次出现南方水稻黑条矮缩病毒(Southern rice black streaked dwarf virus, SRBSDV), 且仅武义县有发病现象。因为该病毒病是一种虫媒病毒, 且白背飞虱Sogatella furcifera (Horváth)是主要传毒介体, 所以本文通过白背飞虱灯下诱虫情况调查、 迁飞轨迹模拟、 天气学背景分析以及毒源地分析, 阐释了2009年浙江省武义县发现的南方水稻黑条矮缩病的供毒源地分布情况, 以及白背飞虱携毒的传递路径, 并讨论了轨迹模拟中各生物学参数的设定方法, 从而为剖析该病毒的宏观流行规律奠定科学基础。结果显示：(1) 通过对白背飞虱迁入武义的主要虫源地与经鉴定的南方水稻黑条矮缩病发病区域的叠加分析, 明确了浙江武义的可能毒源地分布于两广、 闽南、 赣南四省区境内; (2) 西南低空急流及偏南气流是白背飞虱将我国南方的病毒远距离传送到武义县境内的动力源; (3) 白背飞虱随下沉气流和降雨在武义境内的集中降落是南方水稻黑条矮缩病在当地暴发的触发条件。