Fiber chromatographic enabled process intensification increases monoclonal antibody product yield.

The most straightforward method to increase monoclonal antibody (mAb) product yield is to complete the purification process in less steps. Here, three different fiber chromatographic devices were implemented using a holistic approach to intensify the mAb purification process and increase yield. Fiber protein A (proA) chromatography was first investigated, but traditional depth filtration was not sufficient in reducing the contaminant load as the fiber proA device prematurely fouled. Further experimentation revealed that chromatin aggregates were the most likely reason for the fiber fouling. To reduce levels of chromatin aggregates, a chromatographic clarification device (CCD) was incorporated into the process, resulting in single-stage clarification of harvested cell culture fluid and reduction of DNA levels. The CCD clarified pool was then successfully processed through the fiber proA device, fully realizing the productivity gains that the fiber technology offers. After the proA and viral inactivation neutralization (VIN) hold step, the purification process was further intensified using a novel single-use fiber-based polishing anion exchange (AEX) material that is capable of binding both soluble and insoluble contaminants. The three-stage fiber chromatographic purification process was compared to a legacy five-step process of dual-stage depth filtration, bead-based proA chromatography, post-VIN depth filtration, and bead-based AEX chromatography. The overall yield from the five-step process was 60%, while the fiber chromatographic-enabled intensified process had an overall yield of 70%. The impurity clearance of DNA and host cell protein (HCP) for both processes were within the regulatory specification (<100 ppm HCP, <1 ppb DNA). For the harvest of a 2000 L cell culture, the intensified process is expected to increase productivity by 2.5-fold at clarification, 50-fold at the proA step, and 1.6-fold in polishing. Relative to the legacy process, the intensified process would reduce buffer use by 1088 L and decrease overall process product mass intensity by 12.6%.

Tailoring polishing steps for effective removal of polysorbate-degrading host cell proteins in antibody purification.

Ensuring the quality and safety of biopharmaceutical products requires the effective separation of monoclonal antibodies (mAbs) from host cell proteins (HCPs). A major challenge in this field is the enzymatic hydrolysis of polysorbates (PS) in drug products. This study addresses this issue by investigating the removal of polysorbate-degrading HCPs during the polishing steps of downstream purification, an area where knowledge about individual HCP behavior is still limited. We investigated the separation of different mAb formats from four individual polysorbate degrading hydrolases (CES1F, CES2C, LPLA2, and PAF-AH) using cation exchange (CEX) and mixed-mode chromatography (MMC) polishing steps. Our research identified a key challenge: The similar elution behavior of mAbs and HCPs during chromatographic separation. To investigate this phenomenon, we performed high-throughput binding screenings for recombinant polysorbate degrading hydrolases and representative mAb candidates on CEX and MMC chromatography resins. We then employed a three-step strategy that also served as a scale-up process, optimizing separation conditions and leading to the successful removal of specific HCPs while maintaining high mAb recovery rates (>96%). This strategy involved the use of surface response models and miniature columns for screening, followed by validation on larger columns using a chromatography system. Our results highlight the critical role of the inherent properties of mAbs for successful separation from HCPs. These results underscore the need to tailor the purification process to leverage the slight differences in binding behavior and elution profiles between mAbs and specific HCPs. This approach lays the foundation for developing more effective strategies for overcoming the challenge of enzymatic polysorbate degradation, paving the way for improved quality and safety in biopharmaceutical products.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation.

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA.

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone.

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification.

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.

A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics.

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1-2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.

CHO cathepsin B identified as the protease responsible for a target bispecific antibody fragmentation.

In a previous work we demonstrated that CHO protease caused fragmentation of an expressed bispecific antibody (bsAb) and this detrimental host cell protein (HCP) can be effectively removed through an optimized Protein A wash step. In addition, preliminary evidence suggested that the responsible protease belongs to the threonine or cysteine protease family. In the current study, this protease was further identified as cathepsin B. First, we identified several CHO proteases in the further fractionated Protein A wash using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and this allowed us to select four candidate proteases. Next, by examining the cleavage pattern of each individual protease and comparing it with that observed during purification, cathepsin B was identified as the protease responsible for the observed bsAb fragmentation.

Two major mechanisms contributing to copurification of CHO host cell proteins and strategies to minimize their negative impact.

For CHO expressed monoclonal antibodies (mAbs), host cell proteins (HCPs) represent a major class of process-related impurities and their removal is a key focus of downstream process development. HCPs are highly heterogeneous in nature, differing in molecular weight, isoelectric point and hydrophobicity, and some of them can be difficult to remove. Although Protein A affinity chromatography alone can typically remove more than 90% of HCPs in the clarified culture harvest, in many cases reducing HCPs in the final drug product to an acceptable level (e.g., <100 ppm) can still be a challenging task. The relative difficulty of HCP clearance is case dependent and in certain cases a small population of HCPs can persist throughout the downstream process. This article reviews the two major mechanisms that contribute to copurification of CHO HCPs, namely leaching from chromatin heteroaggregates and nonspecific HCP-mAb association. In addition, strategies that can minimize the impact of these two factors are briefly discussed.

Monitoring process-related impurities in biologics-host cell protein analysis.

During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC-MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC-MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC-MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC-MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.

Best practices on critical reagent characterization, qualification, and life cycle management for HCP immunoassays.

The performance of immunoassays for the detection and quantification of host-cell proteins (HCPs) in biopharmaceuticals depends on the quality of the critical assay reagents. Not only their preparation, but also a stringent life-cycle management, including reagent qualification, requalification, and replacement, plays a crucial role in ensuring consistent and reliable results. To provide a cross-industry perspective on HCP reagent management, we conducted a survey on common practices among several pharmaceutical and biotech companies. Based on its outcome, as well as informed by a corresponding roundtable session ("Managing critical reagents over time") at the BioPharmaceutical Emerging Best Practices Association HCP conference in 2019, this study presents specific recommendations and proven concepts to support immunoassay reagent management for monitoring HCPs.

Questioning coverage values determined by 2D western blots: A critical study on the characterization of anti-HCP ELISA reagents.

Host cell proteins (HCPs) constitute a major class of process-related impurities, whose substantial clearance must be demonstrated by suitable analytical methods to warrant product quality and reduce potential safety risks for patients. In this regard, enzyme linked immunosorbent assays (ELISAs), which primarily rely on the quality of the HCP reference standard (immunogen) and HCP-specific polyclonal antibodies, are considered the gold standard for HCP monitoring. For the qualification of the employed antibodies, two-dimensional (2D) western blots (2D-WBs) are the preferred technique to determine the coverage, though a number of practical constraints are well recognized. By using several orthogonal approaches, such as affinity-based mass spectrometry and indirect ELISA, the present study revealed potential detection gaps (i.e., noncovered HCPs) of conventional 2D-WBs, which can be primarily attributed to two different root causes: (i) low amounts of proteins or antibodies being unable to overcome the detection limit and (ii) western blot artifacts due to the loss of conformational epitopes through protein denaturation hindering HCP-antibody recognition. In contrast, the lack of specific antibodies against certain (particularly, low molecular weight) HCPs, as proposed in previous studies, seems to play only a minor role. Together, these findings imply that CHO-HCP ELISA antibodies are better than qualification studies by 2D-WBs indicate.

Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation.

For recombinant proteins produced in Chinese hamster ovary (CHO) cells, fragmentation is a common phenomenon that results in generation of product-related low-molecular-weight (LMW) species. Recently while purifying a bispecific antibody (bsAb), we observed that the target protein experienced cleavage at a couple of potential sites, leading to truncated products. Further studies suggest that the cleavage can likely be attributed to residual CHO cell protease activity. In order to maximally remove potential protease(s) that contribute fragmentation, we optimized Protein A chromatography by adding sodium caprylate (SC) to the wash buffer. Upon optimization, fragmentation of Protein A eluate happened to a much lesser degree as compared to that of eluate from unoptimized process, and the increased sample stability is in accordance with significantly reduced host cell protein (HCP) level. Taken together, the data suggest that SC wash during Protein A chromatography is an effective means for removing HCPs including endogenous protease(s) that are responsible for target antibody fragmentation.

Improved host cell protein analysis in monoclonal antibody products through ProteoMiner.

Host cell proteins (HCPs) impurities are critical quality attributes that have the potential to negatively impact the quality and safety profile of a biopharmaceutical product. Since HCPs often are present at low levels, developing highly sensitive analytical method for their identification and quantitation is critical for process optimization and improvement to reduce them in the final drug product. While an enzyme-linked immunosorbent assay (ELISA) can capture and quantify overall HCP levels, liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool to monitor individual HCP levels during the purification process development. The massive dynamic range of protein species present in a therapeutic antibody is a major challenge for mass spectrometry-based methods to detect low-abundance HCP impurities. This study reports a powerful strategy to identify HCPs in antibody drug substance by applying ProteoMiner enrichment with optimized conditions followed by shotgun proteomic analysis. Using this strategy, we observed that the low abundance HCPs were enriched up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 0.05 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 500 HCPs were confidently identified, which tripled the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were enriched between 100 and 400-fold, highlighting that our method enriches and equalizes all proteins thus improving the sensitivity of HCP identification and quantification.

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., CHOBC ® 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.

Investigation of cathepsin D-mAb interactions using a combined experimental and computational tool set.

Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein-protein interactions. While salt had a moderate effect on the interactions with one of the mAbs, the other mAb demonstrated highly salt-dependent association behavior. Cathepsin D binding to the mAbs was also seen to be highly pH dependent, with operation at pH 9 resulting in a significant decrease in the binding affinity. Protein-protein docking simulations identified three interaction sites on both mAbs; near the complementarity determining region (CDR), in the hinge, and in the CH 3 domain. In contrast, only one face of cathepsin D was identified to interact with all the three sites on the mAbs. Surface property analysis revealed that the binding regions on the mAbs contained strong hydrophobic clusters and were predominantly negatively charged. In contrast, the binding site on cathepsin D was determined to be highly positively charged and hydrophobic, indicating that these protein-protein interactions were likely due to a combination of hydrophobic and electrostatic interactions. Finally, covalent crosslinking coupled with mass spectrometry was used to validate the docking predictions and to further investigate the regions of interaction involved in mAb-cathepsin D binding. A strong agreement was observed between the two approaches, and the CDR loops were identified to be important for cathepsin D interactions. This study establishes a combined experimental and computational platform that can be used to probe mAb-host cell protein (HCP) interactions of importance in biomanufacturing.

Sodium caprylate induced precipitation post Protein A chromatography as an effective means for host cell protein clearance.

In downstream processing of monoclonal antibody (mAb), post Protein A neutralization and subsequent intermediate depth filtration are critical steps for host cell protein (HCP) clearance. Previous studies have shown that adding caprylic acid (CA) during neutralization can further improve HCP removal by promoting their precipitation. In this study, we replaced CA with its sodium salt - sodium caprylate (SC). For the five mAbs studied, SC has been shown to be equally effective as CA at precipitating HCPs. As the salt form has a higher solubility, SC stock solution with relatively high concentration can be easily prepared, which facilitates its adding to the Protein A elution pool. Thus, this study not only confirms the effectiveness of CA/SC-induced HCP precipitation but also provides a more convenient way to integrate this method into the downstream process.

The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance.

Post-load column wash in Protein A chromatography can effectively improve host cell protein (HCP) clearance. A commonly used wash additive for this purpose is sodium chloride. However, the adequate amount of sodium chloride required for effective HCP clearance is less consistent in literature. In this study we investigated the impact of different amounts of sodium chloride on HCP clearance with five monoclonal antibodies (mAbs). For each mAb, elution pool HCP levels from runs under different wash conditions are compared. For all five mAbs, the data suggested that 250 mM would be an adequate amount for the salt to largely achieve its HCP reducing effect. The same conclusion is also reached for calcium chloride, a less commonly used but equally effective Protein A wash additive for HCP clearance.

Comparison of purification strategies for antibodies used in a broad spectrum host cell protein immunoassay.

Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in bulk preparations of biotechnologically produced medicines. Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the polyclonal antibodies (pAbs) used. These pAbs are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. The use of protein A or protein G affinity chromatography results in purified pAbs that are not entirely HCP-specific, while the use of HCP affinity chromatography results in a more specific pAb population but may be harder to recover fully. In theory, both approaches have advantages and disadvantages for generating optimal reagents. In this study, we compared reagents from these two purification procedures using the same starting material, as well as those from a step-wise combination of the two by evaluating purity, concentration, reagent coverage by Western blotting, and performance in an enzyme-linked immunosorbent assay (ELISA). This study demonstrates that pAbs purified by each of the methods are very similar in terms of sensitivity, the ability to recognize a broad range of HCPs, and overall performance in an ELISA measuring a range of HCPs in upstream process and final drug substance (DS) samples.