Application of Advanced Proteomic and Mass Spectrometry Technologies to Enhance the Efficacy of Production of Biopharmaceuticals

Laura Bryan¹ and Paula Meleady^1,2*

• 1 National Institute for Cellular Biotechnology
• 2 School of Biotechnology, Dublin City University, Dublin 9, Ireland
• *Corresponding Author

Abstract

The Chinese Hamster Ovary (CHO) cell line is the dominant mammalian expression system for biopharmaceutical production. Improving the efficiency of production of these biologics will be critical in controlling costs to healthcare systems as more of these drugs come to market. Proteomic and mass spectrometry analysis has been used to profile recombinant CHO cells to get a greater understanding of the molecular and cellular mechanisms that result in a high producing cellular phenotype. In this review we will focus on the application of mass spectrometry (MS) to characterize the proteome of CHO cells and also to delve deeper into the proteome by discussing how mass spectrometry analysis of cellular post translational modifications could potentially play a key role in understanding transcription, growth, cellular signalling events, protein degradation and apoptosis in recombinant CHO cells. Such events are likely to be key to understanding and controlling bioprocess relevant phenotypes to improve the efficacy of production of high quality biotherapeutic proteins.

Introduction

Chinese Hamster Ovary (CHO) cells are known as the workhorses of the biopharmaceutical industry for the production of biotherapeutics.¹ In 2017, the annual sales of biopharmaceuticals were estimated at $188 billion worldwide.² The number of approved products in Europe and the US had steadily increased to 374 in 2018, of which 45 have “blockbuster” status, i.e. sales over $1 billion per year, with monoclonal antibodies (Mabs) representing the most lucrative single product class.² In April 2021, a landmark was reached with the approval by the FDA of the 100th Mab product, GlaxoSmithKline’s PD1 blocker dostarlimab.³ Currently over 84% of Mabs are produced in CHO cells.2 The CHO cell line, therefore, is still the mammalian cell expression system of choice for the production of recombinant therapeutic proteins due to their ability to produce grams/L of high quality protein, the availability of highly effective gene amplification systems, their ability to undertake human-like post-translational modifications (e.g. glycosylation) and protein folding, their robust nature (e.g. conducive to growth in large-scale suspension culture), their track record in industry, and their safety record.^2,4 As a result, there is considerable scientific and commercial interest in research that could improve the efficiency of CHO cell production processes as this dominance is unlikely to change for the foreseeable future.² Within biopharmaceutical companies, the drug discovery pipelines are also producing increasingly complex modalities including fusion proteins, antibody drug conjugates (ADCs), bispecifics, nanobodies and oligomeric structures, which are putting increasing pressure on cell line development groups to generate sufficient, high quality material to meet requirements for clinical trials.⁵

One of the primary aims of pharmaceutical companies is lowering costs associated with development and processes, which makes genetic engineering an appealing area of research as the results can potentially lead to cost reductions. Expression vector improvements have resulted in up to 20-fold improvements in product titers since the 1990s; however, most of these improvements are attributed to optimized feeding strategies and bioprocess regimes. Genetic engineering of CHO cells has become an area of interest amongst researchers in recent years; this is likely due to the publishing of the CHO genome in 2013.⁶ CHO cell lines have also been shown to demonstrate a high degree of heterogeneity6 which can lead to problems in variation on the biopharmaceutical product produced.⁷ This product variation needs to be extensively characterized to ensure the product is safe and efficacious for therapeutic use in patients.

High-producing CHO cell culture processes have been achieved to date using approaches such as optimization of media formulations, improvements in expression vector design and selection systems, and process improvements such as in the design of bioreactors.^8,9 Genetic engineering of the host (CHO) cell itself is also increasingly being used to alter the expression of endogenous genes, in order to improve the efficiency of biotherapeutic protein production.¹⁰ This has been hugely advanced through the development and application of the gene editing tool, CRISPR/Cas,9 to address host- or biopharmaceutical-specific product quality problems. These bioprocessing innovations and cell engineering efforts have improved product titer and product quality; however, uncharacterized cellular processes and gene regulatory mechanisms still hinder cell growth, specific productivity, and protein quality.⁹ In order to enhance the production capabilities and efficiency of the host cell line, an increased understanding of cellular physiology of CHO cells, particularly using advanced ‘omic methods and systems biology, is of critical importance.^9,11 Post-transcriptional bottlenecks suggest there is scope for improvement of secretory pathway machinery in CHO cells. The secretory machinery of CHO cells must also be optimized for the production of more difficult to produce recombinant proteins such as fusion proteins which are understood to be more prone to misfolding.¹²

There are substantial research efforts in progress focusing on systems biology approaches to extensively characterize CHO cell factories. The application of ‘omics-based approaches (e.g. genomics, transcriptomics, proteomics, glycomics, metabolomics, fluxomics, etc.) to CHO cells will enable a fundamental understanding of CHO cell physiology and as a result a better knowledge and understanding of recombinant protein production.⁹ In particular, as the available omics data for CHO cells continue to grow, genomic scale models will be increasingly used to unravel the biological drivers of protein production.¹³ Analysis at the protein level, rather than a the genomic or transcriptomic level, may more directly reflect cellular functions. It has also been shown in many studies that there is a low correlation between gene copy number in the genome and the relative changes at the protein level, meaning that many genomic variations are not or only partially translated to the protein level.¹⁴ In addition, there can be a lack of correlation between mRNA and protein levels, thus examination of the mRNA alone does not necessarily reflect the active cellular functions,¹⁵ hence the importance of studying the proteome in addition to the genome and transcriptome. In addition, proteins are subjected to a variety of post-translational modifications (PTMs) that affect biological activity. The post-translational modification of a protein (e.g. by ubiquitination, phosphorylation, glycosylation, methylation, etc.) is one of the most important mechanisms for regulating protein function by altering activity, cellular location, turnover, and interaction with other proteins;¹⁶ however, alterations to post translational modification of cellular proteins is understudied in recombinant CHO cells.

Proteomic Analyses of CHO Cells Using Mass Spectrometry

Most proteomic studies aim to identify, quantify and characterize expression levels in comparative analysis of proteins. Studies can also focus on determining subcellular location and determining the roles of certain proteins. Top down proteomics is the mass analysis of whole proteins and their isoforms. Advancements in MS technology along with protein chemistry have allowed for the structural analysis of proteins and their complexes, and requires less protein than other structural analysis techniques.¹⁷ Characterization of the protein product itself is a hugely important technique in the biopharmaceutical industry to ensure reproducibility of products between batches.^7,18 Bottom-up proteomics identifies proteins using partial characterization of their amino acid sequence, such as through the use of peptide mapping. In order for this to be possible proteolytic enzyme digestion of intact proteins is carried out before mass spectrometry analysis. Peptides are identified by comparing the masses of proteolytic peptides with predicted proteolytic peptides from a known digested sequence database. Identification of multiple peptide sequences is used for protein identification. Liquid chromatography (LC)-MS/MS analysis requires bioinformatic tools to analyze the raw mass spectral data and convert it into peptide and protein identifications. These bioinformatic tools are used to match large quantities of mass data against predicted masses from protein sequence databases and to perform statistical analysis of samples.

Proteomic profiling of CHO cells themselves can be used to gain an improved understanding of the systems biology of CHO cells, which could enable cellular optimization of bioprocessing.¹⁹ The CHO cell genome was first sequenced in 2011 and reported in 2013.²⁰ Also in recent years, data relating to the proteome, phosphoproteome, glycoproteome and miRNA profile of CHO cells have become available by the collaborative efforts of many researchers.^21–29 The information gathered by these studies has been imperative to advancing the fundamental knowledge of CHO cell biology and understanding what creates advantageous phenotypes.

Quantitative proteomics allows us to measure changes in protein abundance between different types of cells or between the same cells in different conditions, e.g. high recombinant protein producing CHO cells and low recombinant protein producing CHO cells. Quantitative proteomics in combination with LC-MS/MS has become more popular in recent years due to advancements in mass spectrometry instrumentation allowing for the identification of hundreds or thousands of proteins per sample. Methods for both absolute and relative quantitation of peptide abundance have been developed. Proteomic techniques have been used to achieve relative and absolute quantification of CHO cell proteins. Quantitative label free LC-MS/MS methods as well as methods using metabolic and isobaric labels such as Stable Isotope Labeling using Amino Acids in Culture (SILAC), Tandem Mass Tags (TMT) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) have been utilized in the comparison and quantification of proteins in high and low Qp CHO cell lines. For example, iTRAQ labelling has been used to quantitatively identify differentially expressed proteins in high and low Qp (specific productivity) DHFR-GFP fusion protein producing CHO cells and identified an upregulation of proteins associated with protein folding and metabolism.³⁰ SILAC has also been used as a method to investigate the proteome of high producing CHO cell lines after supplementation with sodium butyrate and low temperature culture.³¹ The study found that an increased Qp is associated with sodium butyrate supplementation, and lower temperature culture was associated with an expansion of the cells secretory machinery. Carlage et al. used iTRAQ labeling to show that molecular chaperones such as glucose regulator protein 78 (GRP78) and protein disulfide isomerases were differentially expressed in a Bcl-xL overexpressing mAb producing CHO cell line during the stationary phase of growth.³² More recently, Sommeregger et al. used label-free LC-MS/MS to investigate the proteome of CHO cells expressing two similar single-chain variable fragment antibodies with variations in their thermal stability.³³ They found that CHO cell clones expressing antibody fragments which were less thermally stable also had higher expression of proteins associated with proliferation, apoptosis and cellular stress.

Analysis of Cellular Post Translational Modifications in CHO Cells Using Mass Spectrometry

The vast majority of ‘omic based studies that have been carried out to date in CHO cells have failed to include data on the post-translational level of regulation; however, post translational modifications are crucial to the regulation of many cellular processes including transcription, cytoskeletal rearrangement, cell proliferation, differentiation, apoptosis, protein degradation and signal transduction pathways.

These cellular processes are likely to be hugely important and key to understanding and controlling bioprocess-relevant phenotypes. PTMs determine protein function by altering activity, cellular location, turnover, and interaction with other proteins.¹⁶ 

However, there is little overall work done to date to characterize the role of cellular PTMs in regulating bioprocess-relevant phenotypes, particularly in CHO cells.³⁴ Genomic and transcriptomic approaches are blind to PTMs, making proteomics and LC-MS technologies the only way to study such modifications on a large scale.³⁵ PTMs can be static or dynamic, altering the chemical state of a protein resulting in increased diversity and complexity of the proteome.³⁶ Recent cutting-edge advances in MS based approaches (i.e. high speed, high sensitivity and high resolution) has allowed PTMs to be studied in unprecedented detail in order to evaluate their functional role in the cell.³⁷ 

As previously outlined, there have been a number of proteomic studies in CHO to understanding phenotypes in relation to growth, productivity and product quality by our group and others.^26,38–44 In contrast, very few studies have focused on post-translational modifications on cellular proteins (other than glycosylation 28) in CHO cells, which is surprising given their pivotal role in regulating cellular events such as growth, protein translation and apoptosis.⁴⁵ Our group was first in field to publish global phosphoproteomic analyses of rCHO cells following temperature shift26 and growth phases in batch culture.⁴⁶ In these studies, we have shown specific changes to the phosphorylation status of many proteins of the mTOR and autophagy pathways, 46 pathways which have previously been manipulated in CHO cells to boost growth and productivity.^47–49 These studies show the additional beneficial information generated by including the analysis of cellular PTMs in CHO proteomic studies. Phosphoproteomic analysis also helped show that increased interaction of CREB1 with transgene promoter caused Mab production to be increased in CHO cell lines.⁵⁰ Recently we have shown major changes in the phosphoproteome of industrially relevant high and low productivity IgG4 producing CHO cells.⁴³

Other cellular post translational modifications that have the potential to be more extensively studied in CHO cells include glycosylation, methylation and ubiquitination. For example, ubiquitination involves the conjugation of the 8.5kD protein ubiquitin to target proteins marking them for proteasomal degradation.⁵¹ However, it is becoming increasingly clear that protein ubiquitination does not just target substrates for degradation but also has important regulatory functions similar to phosphorylation, including control of: cell cycle regulation; gene expression; apoptosis; signal transduction; cellular localisation of proteins; and modulating protein-protein interactions.^51,52 Consequently, defects in the ubiquitin pathway are associated with various human diseases including cancer.⁵³ As a result of rapid developments of affinity capture reagents and high-resolution MS it is now feasible to globally analyze the ubiquitinated proteome using quantitative strategies.⁵⁴

Conclusions

Although great improvements have been made to the titer and Qp in CHO cells over the past few decades, we are only at the beginning of developing a true understanding of the intracellular biology of these cells upon which the production of human therapeutic proteins highly relies.

In order to engineer CHO cell lines which are capable of efficiently producing any recombinant protein to a high Qp, we must identify general effector genes. This goal, likely requires a change in the expression of multiple genes, ultimately meaning the targeting of intracellular pathways as opposed to individual genes with individual effects. For this reason, PTMs such as phosphorylation or ubiquitination may present as better potential targets for CHO cell engineering. For example, one phosphorylation event has the potential to trigger an entire signalling pathway. Identifying PTMs which effect bioprocess related phenotypes can lead to the identification of signalling pathways which affect bioprocess related phenotypes. Signalling pathways will likely be more effective targets for CHO cell engineering as these results are less likely to be product or cell line specific. This same principle also applies to miRNAs and transcription factors, which have the ability to alter entire signalling pathways if manipulated. However, targeting entire signalling pathways as opposed to single effector genes does have its disadvantages, such as adverse phenotypic effects from the undesired regulation of a subset of genes.⁵⁵

Acknowledgments

We wish to acknowledge funding from the Irish Research Council Enterprise Partnership Scheme (Project ID EPSPG/2016/10) and from Science Foundation Ireland under Grant number [19/FFP/6759].

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Author Biographies

Laura Bryan is a PhD candidate in CHO cell engineering at the National Institute for Cellular Biotechnology, Dublin City University (DCU) under the supervision of Dr. Paula Meleady. She received an Irish Research Council Enterprise Partnership Scheme award to carry out her research project. She received a BSc in Cell and Molecular Biology in 2015 from University College Dublin, Ireland.

Dr. Paula Meleady is a Principal Investigator at the National Institute for Cellular Biotechnology and an Assistant Professor in the School of Biotechnology, DCU, Ireland. She received her BSc in Biotechnology from DCU in 1993 and a PhD from DCU in 1997. Her research interests are focused on the application of advanced proteomic and mass spectrometry methods to understand biological systems, in particular characterization of recombinant mammalian cell lines (Chinese hamster ovary) in order to gain insights to improving efficiency of production of biopharmaceuticals. She has co-authored over 120 peer-reviewed publications and 12 book chapters to date in research areas related to proteomics, bioprocessing and cancer.

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