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

Modulation of Phosducin-Like Protein 3 (PhLP3) Levels Promotes Cytoskeletal Remodelling in a MAPK and RhoA-Dependent Manner

Background Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in ?-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of ?-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. Methodology/Principal Findings As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of ? and ? tubulin subunits, microtubule disassembly and cell death. In contrast, ?-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. Conclusions Our results support the hypothesis that PhLP3 is important for the maintenance of ?-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth

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

Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins

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

Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production

Please click Additional Files below to see the full abstract

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

No abstract available