Meta-analysis of publicly available Chinese hamster ovary (CHO) cell transcriptomic datasets for identifying engineering targets to enhance recombinant protein yields

Transcriptomics has been extensively applied to the investigation of the CHO cell platform for the production of recombinant biotherapeutic proteins to identify transcripts whose expression is regulated and correlated to (non)desirable CHO cell attributes. However, there have been few attempts to analyse the findings across these studies to identify conserved changes and generic targets for CHO cell platform engineering. Here we have undertaken a meta-analysis of CHO cell transcriptomic data and report on those genes most frequently identified as differentially expressed with regard to cell growth (?) and productivity (Qp). By aggregating differentially expressed genes from publicly available transcriptomic datasets associated with ? and Qp, using a pathway enrichment analysis and combining it with the concordance of gene expression values, we have identified a refined target gene and pathway list whilst determining the overlap across CHO transcriptomic studies. We find that only the cell cycle and lysosome pathways show good concordance. By mapping out the contributing genes we have constructed a transcriptomic ‘fingerprint’ of a high-performing cell line. This study provides a starting resource for researchers who want to navigate the complex landscape of CHO transcriptomics and identify targets to undertake cell engineering for improved recombinant protein output

Exosomes: Biogenesis, targeting, characterization and their potential as "Plug & Play" vaccine platforms

Exosomes are typically characterized as spherical extracellular vesicles less than 150 nm in diameter that have been released into the extracellular environment via fusion of multivesicular bodies (MVBs) to the plasma membrane. Exosomes play a key role in cell-cell communication, vary widely in their composition and potential cargo, and are reportedly involved in processes as diverse as angiogenesis, apoptosis, antigen presentation, inflammation, receptor-mediated endocytosis, cell proliferation, and differentiation, and cell-signaling. Exosomes can also act as biomarkers of health and disease and have enormous potential use as therapeutic agents. Despite this, the understanding of how exosome biogenesis can be utilized to generate exosomes carrying specific targets for particular therapeutic uses, their manufacture, detailed analytical characterization, and methods of application are yet to be fully harnessed. In this review, the author describes the current understanding of these areas of exosome biology from a biotechnology and bioprocessing aspect, but also highlight the challenges that remain to be overcome to fully harness the power of exosomes as therapeutic agents, with a particular focus on their use and application as vaccine platforms

Strategies to control therapeutic antibody glycosylation during bioprocessing: synthesis and separation.

Glycosylation can be a critical quality attribute (CQA) in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity and pharmacokinetics of therapeutic monoclonal antibodies (mAbs) and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering to yield more predictably glycosylated mAbs, and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc gamma-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space in order to establish holistic and consistent manufacturing processes for the production of antibody therapies. This article is protected by copyright. All rights reserved

Defining lncRNAs Correlated with CHO Cell Growth and IgG Productivity by RNA-Seq

How the long non-coding RNA (lncRNA) genome in recombinant protein producing Chinese hamster ovary (CHO) cell lines relates to phenotype is not well described. We therefore defined the CHO cell lncRNA transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs including Adapt15, linked to ER stress, GAS5, linked to mTOR signaling/growth arrest, and PVT1, linked to Myc expression, which are differentially regulated during fed-batch culture and whose expression correlates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc‐fusion protein produced in CHO cells

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins

Eukaryotic elongation factor 2 kinase activity is required for the phenotypes of the Rpl24Bst mouse

No abstract available

Control of translation elongation in health and disease.

Regulation of protein synthesis makes a major contribution to post-transcriptional control pathways. During disease, or under stress, cells initiate processes to reprogramme protein synthesis and thus orchestrate the appropriate cellular response. Recent data show that the elongation stage of protein synthesis is a key regulatory node for translational control in health and disease. There is a complex set of factors that individually affect the overall rate of elongation and, for the most part, these influence either transfer RNA (tRNA)- and eukaryotic elongation factor 1A (eEF1A)-dependent codon decoding, and/or elongation factor 2 (eEF2)-dependent ribosome translocation along the mRNA. Decoding speeds depend on the relative abundance of each tRNA, the cognate:near-cognate tRNA ratios and the degree of tRNA modification, whereas eEF2-dependent ribosome translocation is negatively regulated by phosphorylation on threonine-56 by eEF2 kinase. Additional factors that contribute to the control of the elongation rate include epigenetic modification of the mRNA, coding sequence variation and the expression of eIF5A, which stimulates peptide bond formation between proline residues. Importantly, dysregulation of elongation control is central to disease mechanisms in both tumorigenesis and neurodegeneration, making the individual key steps in this process attractive therapeutic targets. Here, we discuss the relative contribution of individual components of the translational apparatus (e.g. tRNAs, elongation factors and their modifiers) to the overall control of translation elongation and how their dysregulation contributes towards disease processes

Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens