Study of detergent-mediated liberation of hepatitis B virus-like particles from S. cerevisiae homogenate: identifying a framework for the design of future-generation lipoprotein vaccine processes

Virus-like particles (VLPs) are expressed intracellularly in Saccharomyces cerevisiae and the recovery process involves the use of a detergent, which facilitates the release of VLP from host cell components. The detergent-mediated liberation of VLPs is a critical step in primary recovery and is responsible for setting the backdrop for subsequent purification in terms of product yield and characteristics of the process stream. In this paper the use of Triton X-100 detergent for the recovery of lipid envelope VLPs, using the hepatitis B surface antigen (HBsAg) as the VLP model, was investigated. To develop a framework that can be adopted in process design for future generation VLP vaccine candidates, the impact of Triton X-100 was characterized via different response factors: (i) recovery and activity of the HBsAg; (ii) level of protein and lipid contamination from the host cell; and (iii) indirect impact on the performance of an ultrafiltration step following primary recovery. Our studies identified that an increase in detergent concentration favors recovery of HBsAg only to a specific threshold, 0.5% v/v Triton X-100. Further increase in detergent results in delipidation of HBsAg leading to loss in antigenic activity. The level of contamination due to host protein and lipid co-liberation is in proportion with the amount of detergent employed. Greater membrane resistance during ultrafiltration was observed for samples generated using higher concentrations of detergent due to the increase in membrane fouling by the contaminants. Based on this study, Triton X-100 concentrations in the range of 0.2-0.5% v/v appears to be most suitable for recovery of native HBsAg. Choosing between 0.2-0.5% v/v would involve identifying a suitable tradeoff between desired product yield and the level of contamination that can be tolerated by downstream operations.     

An ultra scale‐down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product‐related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell‐based ultra scale‐down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non‐hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (～10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand. Biotechnol. Bioeng. 2013; 110: 1973–1983. © 2013 Wiley Periodicals, Inc.     

Capturing of cell culture‐derived modified Vaccinia Ankara virus by ion exchange and pseudo‐affinity membrane adsorbers

Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300–500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE®, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor‐intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host‐cell DNA levels for human vaccines. This study compares different ion exchange and pseudo‐affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell‐derived MVA‐BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead‐based resin chromatography (Cellufine® sulfate and Toyopearl® AF‐Heparin) was investigated. The two tested pseudo‐affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead‐based column chromatography methods. Overall virus recovery was ～60% for both pseudo‐affinity MA and the Cellufine® sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo‐affinity chromatography materials. Cellufine® sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo‐affinity with anion exchange MA a further reduction of host‐cell DNA was achieved. Biotechnol. Bioeng. 2010. 105: 761–769. © 2009 Wiley Periodicals, Inc.     

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Downstream antibody purification using aqueous two‐phase extraction

The extraction of antibodies using a polyethylene glycol (PEG)‐citrate aqueous two‐phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6‐fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product‐related impurities (aggregates and low molecular product fragments) from ～40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale‐up model for the use of counter‐current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

蛋白A亲和层析法纯化单克隆抗体工艺的优化

治疗性单克隆抗体药物已成为生物医药领域市场最主要的产品类别。蛋白A亲和层析作为第一步捕获抗体蛋白最为有效的手段仍然在现有单克隆抗体纯化平台中占据主导地位。在本研究中,首先开发了一种基于低p H处理抗体细胞回收液的新型细胞液回收技术,该技术能有效去除宿主相关污染物(非组蛋白宿主杂质蛋白、组蛋白、DNA、蛋白聚合物等),同时保证较高的抗体回收率。通过该技术有效预处理后,蛋白A纯化效率可提高10倍左右,并且有效避免了抗体洗脱液中和后浊度的上升,大大减轻了后续蛋白纯化的压力。同时我们也对酸性处理中各种宿主杂质去除机制进行了研究。然后,预处理的洗脱液再经一步Capto adhere色谱纯化,非组蛋白宿主杂质蛋白降低至5 ppm、DNA小于1 ppb、组蛋白降低至检测限以下、蛋白聚合物小于0.01%。总过程抗体蛋白收率87%。该两步法抗体纯化技术可有效集成至当前主流抗体纯化平台,具有良好的大规模应用价值。     

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Single‐chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel?), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere?). The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere? in binding‐mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere? below its theoretical pI, while the majority of HCPs were in the flow‐through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.     

IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ～8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc.     

An important side reaction using the thiol, 3,6‐dioxa‐1,8‐octanedithiol (DODT), in 9‐fluorenylmethoxycarbonyl‐based solid phase peptide synthesis

A considerable quantity of an alkylation by‐product is observed when using 3,6‐dioxa‐1,8‐octanedithiol as a scavenger during acidic release of peptides containing the thioether amino acid methionine from the solid support. Adjustment of the cleavage conditions by replacement of 3,6‐dioxa‐1,8‐octanedithiol with ethane dithiol or by using methionine sulfoxide as an alternative to methionine resulted in no such impurity. The by‐product was detectable by liquid chromatography and mass spectrometry and characterised by NMR spectroscopy of an isolated model peptide. It could be effectively removed in a separate post cleavage step by treatment with dilute aqueous acid at 37 °C. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

A new large‐scale manufacturing platform for complex biopharmaceuticals

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion‐dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non‐ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions. In order to maximize yield, process efficiency, facility and equipment utilization, we have developed, scaled‐up and successfully implemented a new integrated manufacturing platform in commercial scale. This platform consists of a (semi‐)continuous cell separation process based on a disposable flow path and integrated with the upstream perfusion operation, followed by membrane chromatography on large‐scale adsorber capsules in rapid cycling mode. Implementation of the platform at commercial scale for a new product candidate led to a yield improvement of 40% compared to the conventional process technology, while product quality has been shown to be more consistently high. Over 1,000,000 L of cell culture harvest have been processed with 100% success rate to date, demonstrating the robustness of the new platform process in GMP manufacturing. While membrane chromatography is well established for polishing in flow‐through mode, this is its first commercial‐scale application for bind/elute chromatography in the biopharmaceutical industry and demonstrates its potential in particular for manufacturing of potent, low‐dose biopharmaceuticals. Biotechnol. Bioeng. 2012; 109: 3049–3058. © 2012 Wiley Periodicals, Inc.     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high‐throughput small‐scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two‐step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two‐step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:516–520, 2014     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

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.     

Systematic purification of salt‐intolerant proteins by ion‐exchange chromatography: The example of human α‐galactosidase A

Chromatography is an essential tool for purifying biopharmaceutical products. Many processes are still developed based on traditional routines and empirical procedures. Product losses are mostly due to insufficient optimization of purification setups and product sensitivity to process conditions. In order to eliminate these shortcomings, a systematic strategy for the setup of ion‐exchange chromatography is presented, which considers both product stability as well as operational conditions. The stages—a hybrid approach combining high‐throughput screening and analytical small‐scale chromatography—are as follows: (1) pH stability (short‐term); (2) pH stability (long‐term), followed by a screening of additives to enhance protein stability, if required; (3) analytical pH gradient chromatography for evaluation of the operational pH window; and (4) salt stability (long‐term) in the operational pH window determined. The efficiency and straightforwardness of the strategy were shown in a case study on capturing the human α‐galactosidase A enzyme. Following the above procedure, the enzyme was found to be salt‐unstable; a purification factor of 13.2, a concentration factor of 4, and an overall yield of 84.3% were achieved. The applied strategy allowed for a quick establishment of a dedicated capture step at low salt concentrations under stable conditions by well‐chosen prior screening experiments.     

Characterization of the co‐elution of host cell proteins with monoclonal antibodies during protein A purification

Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co‐purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D‐LC‐HDMS^E approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (～10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co‐purification with individual Fc and (Fab′)₂ antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708–717, 2016