Cationic polymer precipitation for enhanced impurity removal in downstream processing

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics.     

Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.     

Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co‐purification of trace levels of ‘hitchhiker’ HCPs during the manufacturing process. Purified mAb‐1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed‐onset particles containing mAb‐1 and its heavy chain C‐terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb‐1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb‐1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP–mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb‐1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb‐1 product. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1360–1369, 2015     

正辛酸沉淀在单克隆抗体纯化中的工艺优化与应用

为应对治疗性抗体快速增长的市场需求,抗体上游细胞培养规模和表达量水平已显著提高,而下游纯化工艺的生产效率则相对落后,下游处理能力已成为限制抗体产能的瓶颈。本研究以单克隆抗体mab-X为实验材料,优化了细胞培养液、低pH病毒灭活收集液2种模式的正辛酸(caprylic acid,CA)沉淀工艺条件,并研究了CA处理去除聚体、CA处理灭活病毒等2种应用,在小试的基础上,采用低pH病毒灭活收集液CA沉淀的模式进行了500 L细胞培养规模生产放大研究,对沉淀前后的产品质量和收率进行了检测和对比。结果显示,两种模式的CA沉淀均可显著降低宿主细胞蛋白(host cell protein,HCP)残留和聚体含量,在聚体去除实验中CA沉淀可去除约15%的聚体,病毒灭活研究显示CA对逆转录模型病毒具有完全的病毒灭活能力。在放大生产规模中,下游依次进行了深层过滤收获、亲和层析、低pH病毒灭活、CA沉淀及深层过滤、阳离子交换层析,CA沉淀过程中混合时间和搅拌速度显著影响CA沉淀效果,CA沉淀处理后低pH病毒灭活液中的HCP残留量降低了895倍,沉淀后产品纯度和HCP残留均已控制在单克隆抗体质量要求范围内,CA沉淀可以减少传统纯化工艺中的一个精纯步骤。总之,下游工艺中采用CA沉淀,能够精简传统纯化工艺,并完全满足mab-X的纯化质量要求,而且能提高生产效率、降低生产成本。本研究结果将推动CA沉淀在单克隆抗体下游纯化生产中的应用,为解决目前传统纯化工艺的问题提供参考。     

ELISA reagent coverage evaluation by affinity purification tandem mass spectrometry

Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been used to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput. There are, however, inherent difficulties reconciling the protein-centric results of MS characterization with ELISA, or providing assurance that ELISA has acceptable coverage against all process-specific HCP impurities that could pose safety or efficacy risks. Here, we describe efficient determination of ELISA reagent coverage by proteomic analysis following affinity purification with a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications can be compared with the actual downstream process impurities for a given process to enable a highly focused assessment of ELISA reagent suitability. We illustrate the utility of this approach by performing coverage evaluation of an anti-HCP polyclonal against both an HCP immunogen and the downstream HCP impurities identified in a therapeutic monoclonal antibody after Protein A purification. The overall goal is to strategically implement affinity-based mass spectrometry as part of a holistic framework for evaluating HCP process clearance, ELISA reagent coverage, and process clearance risks. We envision coverage analysis by AP-MS will further enable a framework for HCP impurity analysis driven by characterization of actual product-specific process impurities, complimenting analytical methods centered on consideration of the total host cell proteome.     

Investigating the combination of single-pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing

There is growing interest within the biopharmaceutical industry to improve manufacturing efficiency through process intensification, with the goal of generating more product in less time with smaller equipment. In monoclonal antibody (mAb) purification, a unit operation that can benefit from intensification is anion exchange (AEX) polishing chromatography. Single-pass tangential flow filtration (SPTFF) technology offers an opportunity for process intensification by reducing intermediate pool volumes and increasing product concentration without recirculation. This study evaluated the performance of an AEX resin, both in terms of host cell protein (HCP) purification and viral clearance, following concentration of a mAb feed using SPTFF. Results show that preconcentration of AEX feed material improved isotherm conditions for HCP binding, resulting in a fourfold increase in resin mAb loading at the target HCP clearance level. Excellent clearance of minute virus of mouse and xenotropic murine virus was maintained at this higher load level. The increased mAb loading enabled by SPTFF preconcentration effectively reduced AEX column volume and buffer requirements, shrinking the overall size of the polishing step. In addition, the suitability of SPTFF for extended processing time operation was demonstrated, indicating that this approach can be implemented for continuous biomanufacturing. The combination of SPTFF concentration and AEX chromatography for an intensified mAb polishing step which improves both manufacturing flexibility and process productivity is supported.     

Real-time monitoring and model-based prediction of purity and quantity during a chromatographic capture of fibroblast growth factor 2

Process analytical technology combines understanding and control of the process with real-time monitoring of critical quality and performance attributes. The goal is to ensure the quality of the final product. Currently, chromatographic processes in biopharmaceutical production are predominantly monitored with UV/Vis absorbance and a direct correlation with purity and quantity is limited. In this study, a chromatographic workstation was equipped with additional online sensors, such as multi-angle light scattering, refractive index, attenuated total reflection Fourier-transform infrared, and fluorescence spectroscopy. Models to predict quantity, host cell proteins (HCP), and double-stranded DNA (dsDNA) content simultaneously were developed and exemplified by a cation exchange capture step for fibroblast growth factor 2 expressed in Escherichia coliOnline data and corresponding offline data for product quantity and co-eluting impurities, such as dsDNA and HCP, were analyzed using boosted structured additive regression. Different sensor combinations were used to achieve the best prediction performance for each quality attribute. Quantity can be adequately predicted by applying a small predictor set of the typical chromatographic workstation sensor signals with a test error of 0.85 mg/ml (range in training data: 0.1–28 mg/ml). For HCP and dsDNA additional fluorescence and/or attenuated total reflection Fourier-transform infrared spectral information was important to achieve prediction errors of 200 (2–6579 ppm) and 340 ppm (8–3773 ppm), respectively.     

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(β-acetylglucosaminyl)- l-asparaginase case study

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(β-acetylglucosaminyl)-l -asparaginase (AGA, EC3.5.1.26) by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS–HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

Polymer‐mediated flocculation of transient CHO cultures as a simple,high throughput method to facilitate antibody discovery

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc‐fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co‐purified during Protein A purification, must be removed in a multi‐step purification process. Our colleagues have reported the use of a novel polymer‐mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel “smart polymer” (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration‐dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393–1400, 2017     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.     

Chromatographic clarification overcomes chromatin-mediated hitch-hiking interactions on Protein A capture column

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHO^BC® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.     

Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process

Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis–sodium dodecyl sulfate (nrCE–SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used during the CE–SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE–SDS versus SDS–PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

A simplified procedure for purification of cytochrome P-450 by preparative ampholine gel for isoelectric focusing

The purification process for cytochrome P450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.     

Host cell protein quantification by fourier transform mid infrared spectroscopy (FT‐MIR)

Process development in up‐ and downstream processing requires enhanced, non‐time‐consuming, and non‐expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP‐enzyme‐linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement. In this work we evaluated the suitability of attenuated total reflection spectroscopy for HCP quantification in process development, using clarified cell culture fluid from monoclonal antibody producing Chinese hamster ovary‐cells after treatment with different polyelectrolytes for semi‐selective clarification. Forty undiluted samples were chosen for multivariate data analysis in the middle infrared range and predicted HCP‐values were in good agreement with results obtained by an ELISA‐assay, suggesting the suitability of this new method for HCP‐quantification. As this method is able to quantify HCP titers ranging from approximately at least 20,000–200,000 ng mL^?1, it is suitable especially for monitoring of process development steps with higher HCP concentrations, omitting dilution errors associated with ELISA assays. Biotechnol. Bioeng. 2013; 110: 252–259. © 2012 Wiley Periodicals, Inc.     

Precipitation of complex antibody solutions: influence of contaminant composition and cell culture medium on the precipitation behavior

Preparative protein precipitation is known as a cost-efficient and easy-to-use alternative to chromatographic purification steps. This said, at the moment, there is no process for monoclonal antibodies (mAb) on the market, although especially polyethylene glycol-induced precipitation has shown great potential. One reason might be the highly complex behavior of each component of a crude feedstock during the precipitation process. For different investigated mAbs, significant variations in the host cell protein (HCP) reduction are observed. In contrast to the precipitation behavior of single components, the interactions and interplay in a complex feedstock are not fully understood yet. This work discusses the influence of contaminants on the precipitation behavior of two different mAbs, an IgG1, and an IgG2. By spiking the mAbs with mock solution, a complex feedstock could successfully be mimicked. Spiking contaminants influenced the yield and purity of the mAbs after the precipitation step, compared to the precipitation behavior of the single components. The mixture showed a decrease in the contaminant and mAb solubility. By re-buffering the mock solution prior to spiking, special salts, small molecules like amino acids, vitamins, or sugars could be depleted while larger ones like HCP or DNA were still present. Therefore, it was possible to distinguish the influence of small molecules and larger ones. Hence, mAb–macromolecular interaction could be identified as a possible reason for the observed higher precipitation propensity, while small molecules of the cell culture medium were identified as solubilisation factors during the precipitation process.

     

Pathogen safety profile of a 10% IgG preparation manufactured using a depth filtration-modified process

Gamunex^®-C is a highly purified liquid 10% IgG preparation manufactured by a process that includes caprylate precipitation and incubation, and chromatography steps. In the original process, caprylate precipitation was followed by cloth filtration to remove impurities. The highly porous cloth filter has since been replaced with a tight depth filter. The impact of this process modification on pathogen reduction and product is presented.Virus and prion reduction was determined under set-point conditions using scaled-down models of the manufacturing process, and at or outside operating limits to determine robustness. Product protein compositions before and after the process modification were compared directly using manufacturing data.Filtration through a tight depth filter substantially increased nonenveloped virus reduction, and virus reduction was maintained even when a compromised depth filter was used. In addition, prion reduction was improved by about three logs. The product IgG content, purity, and IgG subclass distribution remained comparable to the original cloth filtration process.The replacement of cloth filtration with depth filtration increased the pathogen safety margin of the manufacturing process without impacting the product composition.     

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.