Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.

Proteomic analysis of host cell protein fouling during bioreactor harvesting.

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling.

Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.

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.

Ubiquitin: Characterization of a Host Cell Protein Covalently Attached to a Monoclonal Antibody Product by LC-MS/MS.

Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) - MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used.

Immunoinformatic Risk Assessment of Host Cell Proteins During Process Development for Biologic Therapeutics.

The identification and removal of host cell proteins (HCPs) from biologic products is a critical step in drug development. Despite recent improvements to purification processes, biologics such as monoclonal antibodies, enzyme replacement therapies, and vaccines that are manufactured in a range of cell lines and purified using diverse processes may contain HCP impurities, making it necessary for developers to identify and quantify impurities during process development for each drug product. HCPs that contain sequences that are less conserved with human homologs may be more immunogenic than those that are more conserved. We have developed a computational tool, ISPRI-HCP, that estimates the immunogenic potential of HCP sequences by evaluating and quantifying T cell epitope density and relative conservation with similar T cell epitopes in the human proteome. Here we describe several case studies that support the use of this method for classifying candidate HCP impurities according to their immunogenicity risk.

Degradation of polysorbate investigated by a high-performance liquid chromatography multi-detector system with charged aerosol and mass detection.

Polysorbate 80 is widely used as a formulation component in biopharmaceutical drug products. Recent studies have shown that polysorbate 80 is readily degraded either through direct or indirect means. The degradation of polysorbate 80 causes a concern for the long-term stability of biopharmaceutical drug product, as the breakdown products of polysorbate 80 have been shown to cause adverse effects, such as formation of sub-visible and visible particles and mAb aggregation. Understanding the path and extent of degradation is of a paramount importance for the formulator during formulation development. A multi-detector HPLC system using charged aerosol and mass detection was developed and optimized for the characterization of polysorbate 80 standards. The system included a post-column make-up flow, i.e. an inverse gradient, that enabled constant eluent composition at the detectors. The inverse gradient eliminated the main source of variability for the charged aerosol detector response, thereby enabling the calculation of the mass balance between polysorbate components with different degrees of esterification. Extracted ion chromatograms of the mass detector combined with their respective retention times were used to qualitatively characterize the polysorbate samples down to the individual components. The system was applied to study the degradation of several polysorbate standards which occurred by enzymatic digestion or long-term storage. The system provided detailed information on the mechanism of degradation without the need for additional orthogonal analytical techniques.

High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products.

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3MTM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.

Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal.

Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.

Recent applications of quantitative mass spectrometry in biopharmaceutical process development and manufacturing.

Biopharmaceutical products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Aligning with the quality by design (QbD) framework and realization, recent advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation and related techniques have enhanced biopharmaceutical characterization capabilities and have supported an increased development of biopharmaceutical products. Beyond its routine qualitative characterization, the quantitative feature of LC-MS has unique applications in biopharmaceutical process development and manufacturing. This review describes the recent applications and implications of the advancement of quantitative MS methods in biopharmaceutical process development, and characterization of biopharmaceutical product, product-related variants, and process-related impurities. We also provide insights on the emerging applications of quantitative MS in the lifecycle of biopharmaceutical product development including quality control in the Good Manufacturing Practice (GMP) environment and process analytical technology (PAT) practices during process development and manufacturing. Through collaboration with instrument and software vendors and regulatory agencies, we envision broader adoption of phase-appropriate quantitative MS-based methods for the analysis of biopharmaceutical products, which in turn has the potential to enable manufacture of higher quality products for patients.

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry.

Monitoring of host cell proteins (HCPs) during the manufacturing of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug products. Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. Here, we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process. While we also explored spectral library-free DIA interpretation, the spectral library-based approach still showed highest accuracy and reproducibility (coefficients of variation < 10%) with a sensitivity down to the sub-ng/mg mAb level. Thus, this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Quantitation of host cell proteins in biopharmaceuticals from chinese hamster ovarian and vero cell lines using capillary electrophoresis western blots.

Quantitation of host cell proteins (HCPs) is essential in the process of preparation of many biological and vaccine products. Common methods of quantitation include the widely applied enzyme-linked immunosorbent assays (ELISAs), mass spectrometry (MS) and other orthogonal assays. Prior to using these techniques, critical reagents need to be evaluated, for example, antibodies need to be assessed for HCP coverage. Percent of HCP coverage is often established by denatured 2D Western blot. However, ELISAs measure the amount of HCP only in a native state. There are limited studies linking reagents validated by 2D-Western to ensure adequate coverage in the final ELISA. ProteinSimple's newly developed capillary Western blot technology allows for separation, blotting, and detection of proteins in a semi-automated and simplified format. Capillary Westerns are similar to slab Westerns, with the added benefit of being quantitative. Here we outline the capillary Western method that links the 2D Western coverage and ultimately ELISAs for more efficient HCP quantitation. This study describes the development of the capillary Western analytical method to quantitively evaluate HCPs in Vero and Chinese Hamster Ovarian (CHO) cell lines. The amount of CHO HCPs decreases as the sample is purified as expected. Using this approach, we determined that the detected Vero HCPs amount was similar irrespective of denatured (capillary Western) versus native assay format (ELISA). This new method can also be potentially employed to quantitatively assess the anti-HCP antibody reagent coverage used in commercial HCP ELISA kits.

A custom-made time-resolved fluoroimmunoassay for the quantitation of the host cell protein of Vero in rabies vaccine.

Host cell proteins (HCPs) are the process-specific and inevitable impurities during the manufacture via a host cell, which affect the safety or efficacy of the bio-product. However, the commercial HCP enzyme-linked immunosorbent assay (ELISA) kits may not apply to specific products such as rabies vaccine from Vero cells. More advanced and process-specific assay methods are needed in the quality control of rabies vaccine throughout the whole manufacturing process. Therefore, a novel time-resolved fluoroimmunoassay (TRFIA) for the detection of process-specific HCP of Vero cells in rabies vaccine was established in this study. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was used during the preparation of HCP antigen. Based on a sandwich-type immunoassay format, analytes in samples were captured by one antibody coating in the wells and "sandwiched" by another antibody labeled with europium chelates. Due to the complex composition of HCP, both the capture and detected antibodies are polyclonal antibodies from the same anti-HCP antibodies pool. Multiple experiments have identified the optimal conditions to allow the valid and reliable detection of HCP in rabies vaccine. The TRFIA had a satisfactory limit of detection value (0.011 µg/ml) under optimal conditions, with the linear range from 0.0375 to 2.4 µg/ml of HCP. The coefficient variations (CVs) were all < 10%, and the recoveries were in the range of 97.00-102.42%. All the test results of Vero cell protein reference substance were included in the expected concentration, which demonstrated that the present method was available for the test of HCP in rabies vaccine. Based on these results, the novel TRFIA to detect HCP appears to be important for application in modern vaccine quality control during the whole manufacturing process.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Comparison of host cell protein removal by depth filters with diatomaceous earth and synthetic silica filter aids using model proteins.

A number of studies have demonstrated that depth filtration can provide significant adsorptive removal of host cell proteins (HCP), but there is still considerable uncertainty regarding the underlying factors controlling HCP binding. This study compared the binding characteristics of two fine grade depth filters, the X0SP (polyacrylic fiber with a synthetic silica filter aid) and X0HC (cellulose fibers with diatomaceous earth (DE) as a filter aid), using a series of model proteins with well-defined physical characteristics. Protein binding to the X0SP filter was dominated by electrostatic interactions with greatest capacity for positively-charged proteins. In contrast, the X0HC filter showed greater binding of more hydrophobic proteins although electrostatic interactions also played a role. In addition, ovotransferrin showed unusually high binding capacity to the X0HC, likely due to interactions with metals in the DE. Scanning Electron Microscopy with Energy Dispersive Spectroscopy was used to obtain additional understanding of the binding behavior. These results provide important insights into the physical phenomena governing HCP binding to both fully synthetic and natural (cellulose + DE) depth filters.

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.

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing.

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.

Two-Dimensional Gel Electrophoresis and 2D-DIGE.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system, particularly in the separation of intact proteins. A modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual protein species including protein isoforms and post-translational modifications. This chapter discusses the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research, particularly on recombinant Chinese hamster ovary cells, which are also discussed in this chapter.

Methods for addressing host cell protein impurities in biopharmaceutical product development.

The high demand for monoclonal antibody (mAb) therapeutics in recent years has resulted in significant efforts to improve their costly manufacturing process. The high cost of manufacturing mAbs derives mainly from the purification process, which contributes to 50%-80% of the total manufacturing cost. One of the main challenges facing industry at the purification stage is the clearance of host cell proteins (HCPs) that are produced and often co-purified with the desired mAb product. One of the issues HCPs can cause is the degradation of the final mAb protein product. In this review, techniques are considered that can be used at different stages (upstream and downstream) of mAb manufacture to improve HCP clearance. In addition to established techniques, many new approaches for HCP removal are discussed that have the potential to replace current methods for improving HCP reduction and thereby the quality and stability of the final mAb product.