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Abstract The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry‐wide consortium collaborated to develop a study to characterize age‐dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)‐producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO‐K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two‐dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age‐dependent expression were previously reported to be problematic as high‐risk and/or difficult‐to‐remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical. 

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing. 

Chinese hamster ovary (CHO) cells are the primary mammalian cell lines utilized to produce monoclonal antibodies (mAbs). The upsurge in biosimilar development and the proven health benefits of mAb treatments reinforces the need for innovative methods to generate robust CHO clones and enhance production, while maintaining desired product quality attributes. Among various product titer-enhancing approaches, the use of histone deacetylase inhibitors (HDACis) such as sodium butyrate (NaBu) has yielded promising results. The titer-enhancing effect of HDACi treatment has generally been observed in lower producer cell lines but those studies are typically done on individual clones. Here, we describe a cell line development (CLD) platform approach for creating clones with varying productivities. We then describe a method for selecting an optimal NaBu concentration to evaluate potential titer-enhancing capabilities in a fed-batch study. Finally, a method for purifying the mAb using protein A chromatography, followed by glycosylation analysis using mass spectrometry, is described. The proposed workflow can be applied for a robust CLD process optimization to generate robust clones, enhance product expression, and improve product quality attributes. 

Host cell protein (HCP) impurities, endogenous proteins expressed from host cells, can challenge biopharmaceutical manufacturing. Certain HCPs can persist even after downstream purification, leading to adverse impacts on drug stability and potentially, patient safety. Thus, the quantification and control of HCPs is critical. Although many improvements have been made in HCP quantification and control methods, HCP-associated risks cannot be completely eliminated. A better biophysical understanding of Chinese hamster ovary (CHO) HCPs and advancement of monitoring assays will lead to better controlled biopharmaceutical manufacturing. This chapter will discuss (i) current HCP removal processes for various product types, (ii) the impact of residual HCPs on drug efficacy and safety, (iii) HCP quantification and monitoring methods such as proteomics approaches and enzyme-linked immunosorbent assays (ELISA) using anti-HCP antiserum, (iv) HCP control approaches in both upstream and downstream processes, and (v) future directions for effective HCP risk management strategies.