Paula Meleady

Objective Chinese hamster ovary (CHO) cells are the dominant platform for monoclonal antibody (mAb) production. Despite their widespread use, an understanding of endoplasmic reticulum (ER) stress during recombinant protein production remains limited, often creating bottlenecks that hinder improvements in production titres and product quality. Because many critical regulators of protein folding, processing and quality control are membrane-embedded or membrane-associated, they are frequently underrepresented in whole-cell lysate proteomic analyses. In this study, we applied a membrane proteome enrichment strategy to selectively enhance detection of ER-resident and secretory pathway proteins and thereby improve the resolution of ER stress responses in recombinant CHO cell lines. Methods Sustained ER stress was induced using tunicamycin for 72 h in two IgG producing CHO cell lines, CHO DP-12 and NISTCHO. Membrane-enriched proteomes were analysed using label-free quantitative LC–MS/MS, followed by orthogonal validation using targeted LC–MS. Results Membrane enrichment expanded coverage of ER-resident and secretory pathway proteins, revealing minimal overlap with whole-cell lysate analyses. ER stress induced distinct, pathway-level changes in the two cell lines that were more clearly resolved in the membrane fraction. In CHO DP‑12 cells, ER stress resulted in decreased abundance of proteins involved in protein folding, oxidative folding, and glycosylation, together with altered abundance of proteins involved in ER-associated degradation (ERAD) suggesting remodelling of the ER quality-control network and a potential shift towards enhanced clearance of misfolded proteins. In contrast, NISTCHO cells showed more moderate changes, including increased abundance of several ER chaperones and oxidative folding proteins, alongside comparatively limited changes in degradation-related pathways. Targeted LC–MS using parallel reaction monitoring (PRM) confirmed the direction of regulation of a panel of these proteins, providing additional confidence in the observed trends. Conclusions This study highlights membrane proteomics as an important approach for studying ER-associated processes relevant to CHO cell function under ER stress conditions.

Chinese hamster ovary (CHO) cells are widely utilised in the biopharmaceutical industry to produce therapeutic proteins. Understanding the mechanisms of endoplasmic reticulum (ER) stress and its interplay with protein degradation pathways remains pivotal for improving production efficiency and product quality. In this study, we investigated the proteomic responses of CHO-K1 (non-producer), CHO DP-12 (IgG-producer), and NISTCHO (IgG-producer) cell lines under ER stress induced by a combination of the proteasome inhibitor MG132 and the glycosylation inhibitor tunicamycin. Viability, cell growth, and IgG titre were measured after 24 h, 48 h, and 72 h of treatment and the 48 h timepoint was used for the comparative analysis of the proteomic data across the three cell lines. Proteasome inhibition with MG132 intensified ER stress and altered ER-associated protein degradation (ERAD). Combined tunicamycin + MG132 treatment was associated with cell line-specific proteomic changes: NISTCHO upregulated ER translocation and glycoprotein quality control proteins (SSR4, SEC24C, UGGT1), CHO DP-12 activated redox/disulfide regulators (DNAJC10, CAPN1), while CHO-K1 showed broad proteome shifts, suggesting differences in baseline stress handling. These findings provide mechanistic insights into ER stress and protein quality control in CHO cells, offering a foundation for strategies to enhance cell line robustness and optimise biopharmaceutical production.

Chinese hamster ovary (CHO) cells, widely utilised in biopharmaceutical production, experience various stressors during cell culture that can affect protein expression and folding, particularly within the endoplasmic reticulum (ER). Mild hypothermia is widely employed in CHO cell bioproduction to improve recombinant protein yield and quality; however, its impact on ER‐associated pathways, particularly those governing protein folding and stress responses, remains insufficiently characterised. Mass spectrometry‐based proteomics allows for the identification and relative quantification of proteins, enabling detailed insights into protein expression, modifications, and functional networks. This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of CHO cells, with a specific focus on ER proteins and ER stress. Using high‐resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia. The downregulation of several proteins involved in the glycosylation of nascent polypeptides at 31°C, including DDOST, P4HB, PRKSCH and LMAN1, in all cell lines studied suggests that mild hypothermic shock disrupts the cell's normal ability to fold new proteins, leading to ER stress as the misfolded proteins build up. When this is coupled with the maintained cell viability and increased productivity at 31°C, it indicates the ER stress response can mitigate the build‐up of misfolded proteins. The differential regulation of the transcription factor eIF2α, downregulated in non‐producer cells but upregulated in producer cells at 31°C, suggests that recombinant protein‐producing CHO cells possess a more adaptive ER stress response, enabling more efficient function under hypothermic culture conditions. Enhanced ubiquitination of misfolded protein substrates highlights an increased reliance on ER‐associated degradation (ERAD) pathways to alleviate proteotoxic stress, as well as the wide range of biological processes that are regulated by ubiquitination as part of the hypothermic stress response. These findings provide new insights into the cellular adaptation mechanisms of CHO cells to mild hypothermia, with implications for optimising bioproduction strategies to improve yield and quality of therapeutic proteins. Our study highlights the importance of understanding the more complex aspects of the proteome and how this additional layer of detail can open new avenues for CHO cell engineering.

It is well established that the biomolecular corona affects the biological behavior of nanomaterials, including cellular uptake, toxicity, and biodistribution. However, the unique physicochemical properties of advanced materials, such as graphene oxide, challenge the effectiveness of standard protocols for biomolecular corona characterization, which may lead to incomplete biomolecule recovery and biased experimental results. Protein analysis is one of the broadest techniques in the characterization of the biomolecular corona, providing important information about the composition and behavior of proteins adsorbed onto nanomaterial surfaces. Two widely accepted protein analysis methods include SDS-PAGE and mass spectrometry, and both require the complete elution of the proteins from the nanoparticle surface during denaturation steps. In this work, limitations of widely used SDS-based elution methods with GO were identified, and an improved protocol using chaotropic agents (urea and thiourea) was developed. The stepwise extraction allowed for near-complete protein desorption. Under the modified protocol, strongly bound proteins that are more hydrophobic have been proved to be underestimated using the standard method. This further reiterates the necessity for the development of methodologies tailored to the specific materials under study which accurately characterize the protein corona. Our results highlight the need for standardization and optimization of protocols to ensure reproducibility and reliability in nanosafety studies, hence promoting the safe and sustainable use of advanced materials in biological and environmental systems.

Dihydrofolate reductase activity is required in One Carbon Metabolism to ensure that the biologically active form of folate, tetrahydrofolate, is replenished and available as an enzyme cofactor for numerous cellular reactions, including purine and pyrimidine synthesis. Most cellular enzyme activity was thought to arise from the product of the DHFR gene on chromosome 5, with its paralogue DHFR2 (formerly known as DHFRL1; [chromosome 3]), believed to be responsible for mitochondrial dihydrofolate activity based on recombinant versions of the enzyme. In this paper, we confirm our earlier findings that dihydrofolate reductase activity in mitochondria is derived from the DHFR gene rather than DHFR2 and that endogenous DHFR2 protein is not detectable in most cells and tissues. Using HepG2 cell lines with modulated expression of either DHFR or DHFR2, we observed an impact of DHFR2 RNA on One Carbon Metabolism mediated through an influence on DHFR expression and activity. Knockout of DHFR2 results in a drop in dihydrofolate reductase activity, lowered 10‐formyltetrahydrofolate abundance, downregulation of DHFR mRNA, and diminished DHFR protein abundance. We also observed downregulation of Serine Hydroxymethyltransferase and Thymidylate Synthase, two One Carbon Metabolism enzymes that work with DHFR to support de novo thymidylate synthesis. The expression of recombinant DHFR2 resulted in restoration of DHFR mRNA and protein levels while a DHFR knockdown cell line showed upregulation of DHFR2 RNA. We propose that the DHFR2 gene encodes an RNA molecule that regulates cellular dihydrofolate reductase activity through its impact on DHFR mRNA and protein.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER‐stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC‐MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO‐DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N‐glycan biosynthesis, and ubiquitin‐mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER‐stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies.

Identifying and quantifying host cell proteins (HCPs) are crucial in developing and manufacturing biopharmaceutical products. ELISA has been used widely for identifying and quantifying HCPs in biopharmaceutical products. Liquid chromatography-mass spectrometry (LC-MS) has recently emerged as an orthogonal tool for HCP analysis, providing comprehensive and quantitative information on individual HCPs. This chapter explores the advancements in LC-MS/MS methodologies for HCP analysis, including sample preparation, data acquisition modes, and data analysis strategies. It also discusses the challenges of HCP identification and quantitation, and recent innovations to overcome these limitations. The application of LC-MS/MS methods in HCP profiling holds significant promise for improving process control and ensuring product safety in biopharmaceutical manufacturing.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.