Design of salt‐tolerant membrane adsorbers for viral clearance

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50–150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage ΦX174 (pI ～ 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand‐binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance‐processing step compared to traditional strong anion exchange membrane adsorbers. Biotechnol. Bioeng. 2009;103: 920–929. © 2009 Wiley Periodicals, Inc.     

Viral clearance by flow‐through mode ion exchange columns and membrane adsorbers

Anion exchange (AEX) is a common downstream purification operation for biotechnology products manufactured in cell culture such as therapeutic monoclonal antibodies (mAbs) and Fc‐fusion proteins. We present a head‐to‐head comparison of the viral clearance efficiency of AEX adsorbers and column chromatography using the same process fluids and comparable run conditions. We also present overall trends from the CDER viral clearance database. In our comparison of multiple brands of resins and adsorbers, clearance of three model viruses (PPV, X‐MuLV, and PR772) was largely comparable, with some exceptions which may reflect run conditions that had not been optimized on a resin/membrane specific basis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:124–131, 2014     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

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     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.     

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part II. Virus,host cell protein,DNA clearance,and antibody recovery

Anion exchange membrane adsorbers are used for contaminant removal in flow‐through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion‐exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0–200 mM NaCl, pH 6.0–9.0, and flow rates of 4–20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion‐exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow‐through polishing steps. Biotechnol. Bioeng. 2013; 110: 500–510. © 2012 Wiley Periodicals, Inc.     

Impact of antibody aggregation on a flowthrough anion‐exchange membrane process

The impact of typical anion‐exchange flowthrough conditions on the IgG mass loading of an anion‐exchange membrane scale‐down unit (Mustang® Q coin) was investigated. High performance size‐exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion‐exchange flowthrough conditions. The small filtration area presented by the 0.35 mL membrane volume Mustang® Q coin limited the membrane throughput due to fouling from the aggregates at higher antibody loading. Data in this report indicated that a 0.2 μm hybrid polyethersulfone and polyvinylidene fluoride membrane in‐line prefilter with a minimum filtration area of 20 sq cm alleviated the Mustang® Q coin fouling. The combined cake filtration and intermediate blocking model was proposed as the most likely membrane pore blocking mechanism. Increasing the filtration area in the in‐line prefilter resulted in higher IgG mass throughput. Thus, using an appropriately sized in‐line prefilter could provide more robust antibody throughput performance on scale‐down membrane anion‐exchange units. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

Effects of pH,conductivity, host cell protein,and DNA size distribution on DNA clearance in anion exchange chromatography media

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process‐related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ‐650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141–149, 2018     

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non‐infectious retrovirus‐like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO^? and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV. Biotechnol. Bioeng. 2012;109: 157–165. © 2011 Wiley Periodicals, Inc.     

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.     

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.     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.     

Novel spiking methods developed for anion exchange chromatography operating in a continuous process

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in-line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow-through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing.     

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture‐based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu®) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine® sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture‐derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell‐derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM‐adsorbers were compared directly to column‐based Cellufine® sulfate and commercially available cation‐exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM‐capture step resulted in a higher reduction of dsDNA compared to the tested cation‐exchange membrane adsorbers. The productivity of the SCM‐based unit operation could be significantly improved by a 30‐fold increase in volumetric flow rate during adsorption compared to the bead‐based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell‐derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM‐adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine® sulfate. Biotechnol. Bioeng. 2009;103: 1144–1154. © 2009 Wiley Periodicals, Inc.     

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 log₁₀ 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. Biotechnol. Bioeng. 2010;106: 238–246. Published 2010 Wiley Periodicals, Inc.     

Development of a 3‐step straight‐through purification strategy combining membrane adsorbers and resins

The pressures to efficiently produce complex biopharmaceuticals at reduced costs are driving the development of novel techniques, such as in downstream processing with straight‐through processing (STP). This method involves directly and sequentially purifying a particular target with minimal holding steps. This work developed and compared six different 3‐step STP strategies, combining membrane adsorbers, monoliths, and resins, to purify a large, complex, and labile glycoprotein from Chinese hamster ovary cell culture supernatant. The best performing pathway was cation exchange chromatography to hydrophobic interaction chromatography to affinity chromatography with an overall product recovery of up to 88% across the process and significant clearance of DNA and protein impurities. This work establishes a platform and considerations for the development of STP of biopharmaceutical products and highlights its suitability for integration with single‐use technologies and continuous production methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:931–940, 2017     

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: part II. DNA and virus separations

The surface-initiated polymerization protocol developed in part I was used to prepare strong anion-exchange membranes with variable polymer chain graft densities and degrees of polymerization for DNA and virus particle separations. A focus of part II was to evaluate the role of polymer nanolayer architecture on DNA and virus binding. Salmon sperm-DNA (SS-DNA) was used as model nucleic acid to measure the dynamic-binding capacities at 10% breakthrough. The dynamic-binding capacity increases linearly with increasing poly ([2-(methacryloyloxy)ethyl]trimethylammonium chloride) chain density up to the highest chain density used in this study. The new membranes yielded threefold higher SS-DNA-binding capacity (30 mg/mL) than a leading commercial membrane with the same functional group chemistry. Elution of bound DNA yielded a sharp peak, and resulted in a 13-fold increase relative to the feed concentration. This concentration effect further demonstrates the highly favorable transport properties of the newly designed Q-type membranes. However, unlike findings in part I on protein binding, SS-DNA binding was not fully reversible. Minute virus of mice (MVM) was used as model virus to evaluate the virus clearance performance of newly designed Q-type membranes. Log reduction of virus (LRV) of MVM increased with increasing polymer chain density. Membranes exhibited >4.5 LRV for the given MVM impurity load and may be capable of higher LRV values, as the MVM concentration in the flow-through fraction of these samples was below the limit of detection of the assay.     

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