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Conflict of interest statement
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as
Introduction Over the past decade, more than a hundred new biopharmaceutical products have been approved and marketed in the United States (US) and European Union (EU). Market value for these biologics was recently estimated at $140 billion US, with a total of over two hundred therapeutics (Walsh, 2014). A significant portion of these products are recombinant proteins, with an ongoing increase in the number of them produced in mammalian expression platforms (Walsh, 2014). This trend is mostly driven by the increased attention directed to post-translational modifications of these biologics, in particular towards their glycosylation state. Indeed, several efforts have been made over the last few years to understand how glycosylation can influence the biological activity of therapeutics. Studies have demonstrated that proper glycosylation profiles can improve recombinant protein properties such as increase their stability and half-life in blood circulation and decrease their immunogenicity (Ashwell and Harford, 1982, Runkel et al., 1998, Ghaderi et al., 2010, Ghaderi et al., 2012, Jefferis, 2016a, Jefferis, 2016b, Kuriakose et al., 2016). Among the mammalian-based expression systems, CHO cells is by far, the most commonly used cell line. It is involved in the production of over 70% of recombinant biopharmaceutical proteins, most of them being monoclonal INCB3344 synthesis (mAbs) (Durocher and Butler, 2009, Kim et al., 2012, Butler and Spearman, 2014). This review will summarize the recent advances in production of glycoproteins in mammalian cells, with a particular emphasis on the CHO cell system. The various expression systems currently used for therapeutic glycoprotein production (Fig. 1) will be overviewed and cell engineering strategies used to improve biologics production and/or quality will be discussed. Finally, we will also describe the different “omics” approaches used lately in the field in order to improve glycoprotein production and/or glycosylation.
Cell engineering In the biopharmaceutical industry, production of glycoproteins is currently achieved by either transient or stable gene expression in the cells. When the need for a quick and economical approach prevails, transient expression remains the best choice for protein production (Rosser et al., 2005, Pham et al., 2006, Geisse, 2009, Chahal et al., 2011, Baldi et al., 2012). By skipping the lengthy selection process for the cells that have integrated the plasmid within their genome, transient transfection is much faster (Rita Costa et al., 2010). However, the production rate relies on many factors including efficiency of the transfection phase, cytotoxicity of the transfection reagent and extensiveness of the feeding strategy applied to the culture post-transfection (Pham et al., 2006, Daramola et al., 2014). Although the level of protein obtained is not as high as with stable gene expression, it is still sufficient for many applications. Indeed, during transient transfection, the plasmid DNA is mostly kept extrachromosomally, the cells rapidly losing it during division, therefore limiting the amount of expressed proteins. Yet, it is ideal for high throughput screening for hits identification and very helpful for early stage product characterization (Ozturk and Hu, 2005, Rosser et al., 2005, Rita Costa et al., 2010). To date, only viral vectors used in gene therapy have been produced by transient transfections for clinical applications (Wright, 2009). When it comes to production of glycoproteins in large quantities, stable gene expression systems remain the preferred avenue. For these systems, multiple aspects have been tackled and optimized for improving productivity, process robustness and reducing cell line generation timelines (Fig. 2).