Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography.

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure-property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

Impact of ligand structure and base bead pore size on host cell protein removal during monoclonal antibody purification using multimodal chromatography resin.

Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.

In-house CHO HCPs platform: A promising approach for HCPs ELISA monitoring.

A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities. Residual host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme-linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts. This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.

Risk control of host cell proteins in one therapeutic antibody produced by concentrated fed-batch (CFB) mode.

Multiple control strategies, including a downstream purification process with well-controlled parameters and a comprehensive release or characterization for intermediates or drug substances, were implemented to mitigate the potential risk of host cell proteins (HCPs) in one concentrated fed-batch (CFB) mode manufactured product. A host cell process specific enzyme-linked immunosorbent assay (ELISA) method was developed for the quantitation of HCPs. The method was fully validated and showed good performance including high antibody coverage. This was confirmed by 2D Gel-Western Blot analysis. Furthermore, a LC-MS/MS method with non-denaturing digestion and a long gradient chromatographic separation coupled with data dependent acquisition (DDA) on a Thermo/QE-HF-X mass spectrometer was developed as an orthogonal method to help identify the specific types of HCPs in this CFB product. Because of the high sensitivity, selectivity and adaptability of the new developed LC-MS/MS method, significantly more species of HCP contaminants were able to be identified. Even though high levels of HCPs were observed in the harvest bulk of this CFB product, the development of multiple processes and analytical control strategies may greatly mitigate potential risks and reduce HCPs contaminants to a very low level. No high-risk HCP was identified and the total amount of HCPs was very low in the CFB final product.

Immunoreactivity profiling of Anti-Chinese hamster ovarian host cell protein antibodies by isobaric labeled affinity purification-mass spectrometry reveals low-recovery proteins.

We evaluated the immunoreactivity profiles of eight commercial anti-host cell protein (anti-HCP) antibodies from different host animals and their antigens used for immunization by an isobaric labeled affinity purification-mass spectrometry (AP-MS) method. As a result, 34 proteins with high abundance but low recovery from harvest cell culture fluid were identified. Since they are likely to be underestimated in biopharmaceutical quality assessment, the features common to these proteins were investigated. Compared to other immunoprecipitated HCP proteins, proteins exhibiting lower molecular weight (ΔMW = -14600), lower isoelectric point (ΔpI = -0.86), and lower hydrophobicity (ΔGRAVY = -0.13) were enriched. This AP-MS method provides important information for HCP control strategies using immunological methods and is expected to contribute to the development of safe biopharmaceutics.

Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.

Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated. It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to ˂ 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.

Targeted CHO cell engineering approaches can reduce HCP-related enzymatic degradation and improve mAb product quality.

Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification.

Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

Optimized Sample Preparation and Data Processing of Data-Independent Acquisition Methods for the Robust Quantification of Trace-Level Host Cell Protein Impurities in Antibody Drug Products.

Host cell proteins (HCPs) are a major class of bioprocess-related impurities generated by the host organism and are generally present at low levels in purified biopharmaceutical products. The monitoring of these impurities is identified as an important critical quality attribute of monoclonal antibody (mAb) formulations not only due to the potential risk for the product stability and efficacy but also concerns linked to the immunogenicity of some of them. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS)-based approaches have been emerging as powerful and promising alternatives providing qualitative and quantitative information. However, a major challenge for liquid chromatography (LC)-MS-based methods is to deal with the wide dynamic range of drug products and the extreme sensitivity required to detect trace-level HCPs. In this study, we developed powerful and reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, the library-free data-independent acquisition (DIA) method, and stringent validation criteria. The performances of several preparation protocols and DIA versus classical data-dependent acquisition (DDA) were evaluated using a series of four commercially available drug products. Depending on the selected protocols, the user has access to different information: on the one hand, a deep profiling of tens of identified HCPs and on the other hand, accurate and reproducible (coefficients of variation (CVs) < 12%) quantification of major HCPs. Overall, a final global HCP amount of a few tens of ng/mg mAb in these mAb samples was measured, while reaching a sensitivity down to the sub-ng/mg mAb level. Thus, this straightforward and robust approach can be intended as a routine quality control for any drug product analysis.

Aqueous Two-Phase-Assisted Precipitation of Proteins: A Platform for Isolation of Process-Related Impurities from Therapeutic Proteins.

Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components. Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids. This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250-500 to 10-15 ppm) and of nucleic acids (from 15-20 to 5-15 ng/mL) were observed.

Sensitive, Rapid, Robust, and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development.

There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels. In some cases, regulatory agencies require the identification and the quantification of HCPs in drug products (DPs) for risk assessment, and this is an active and growing topic of conversation in the industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in a significantly shorter turnaround time than that previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Because of its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances and DPs.

Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies.

The repertoire of complex proteins produced by the host cell during monoclonal antibody (mAb) production has generated a bottleneck in downstream bioprocessing. Low ppm levels of host cell proteins (HCPs) must be achieved at the downstream purification process stage to generate an end product suitable for use in humans. The increased demand for mAb drug products globally has driven research to focus on affordability of mAb production platforms. This has fuelled advancements in manufacturing R&D to deliver higher product titres with better economics without sacrificing product quality. This study highlights the beneficial effects of inclusion of the Emphaze™ AEX Hybrid Purifier, compared to a conventional clarification process, for removal problematic HCPs during downstream bioprocessing of mAbs. Advanced proteomic methods were used to track and identify known 'problematic' HCPs through a multi-cycle Protein A purification process. Removal of histone proteins was observed, along with an average total HCP reduction of 38-fold and an average reduction of 2.3 log in HCDNA concentration. Chromatographic clarification using the Emphaze™ AEX Hybrid Purifier in conjunction with Protein A chromatography resulted in the removal of problematic HCPs including 78 kD glucose-regulated protein, nidogen-1, heat shock proteins, actin, serine protease HTRA1 and matrix metalloproteinase-19. It is shown herein that the Emphaze™ AEX Hybrid Purifier, which is readily incorporated into a mAb purification process during the clarification stage, has the potential to increase Protein A resin lifetime and potentially reduce the number of subsequent polishing chromatographic steps needed to remove HCPs that have a tendency to co-purify with mAb products.

The application of caprylic acid in downstream processing of monoclonal antibodies.

Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used as albumin stabilizer, non-IgG fraction precipitant and bactericidal agent in pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule's protein/lipid binding capacity conferred by its octyl moiety. This article, following an initial review of CA's historical applications, introduces CA's relatively new application in downstream processing of monoclonal antibodies (mAbs). By taking advantage of CA mediated impurity precipitation and virus inactivation, it might be possible to develop a two-column purification process in replacement of the standard three-column process without compromising product quality.

Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses.

Host cell protein content during bioprocessing of biotherapeutic proteins generated from cultured Chinese hamster ovary (CHO) cells is typically measured using immunological and gel-based methods. Estimation of HCP concentration is usually undertaken using Enzyme-Linked ImmunoSorbent Assays (ELISA), while estimation of HCP clearance/presence can be achieved by comparing 2D-PAGE images of samples and by undertaking western blotting of 2D-PAGE analyzed samples. Here, we describe the analyses of HCP content using these methodologies.

Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins.

Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product. Hence reducing them to the generally accepted level can be challenging. This article reviews effective HCP removing strategies at different stages of downstream process for monoclonal antibodies and Fc-fusion proteins. When used in combination, these strategies can greatly enhance the chance of meeting the drug substance specifications for residual HCP.

A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products.

This article presents a variant of aqueous two phase system (ATPS) as a tool for selective removal of process related impurities associated with Escherichia coli, namely host cell proteins and nucleic acids. Granulocyte colony stimulating factor (GCSF) expressed in E. coli has been selected as a model protein for the study. While achieving effective removal of host cell impurities as per the regulatory requirement for recombinant therapeutics, high product recovery has been achieved by adopting a novel strategy involving resolubilization of interfacial GCSF precipitate. This has been done such that the structural and biological activity of the product is retained. Exhaustive analysis of structural as well as functional integrity of resolubilized GCSF has been carried out using advanced analytical and in vitro bioassay tools. Product recovery of 99.5% has been achieved with the concentration of host cell proteins less than 100ppm and of nucleic acids below 10ng/ml. We think that the proposed platform can enable use of ATPS as a more economical alternative to process chromatography in industrial biopharmaceutical manufacturing processes.