Protein Glycosylation: Analysis, Characterization, and Engineering

Abstract

Protein glycosylation is critically important in vivo; current estimates are that more than half of the proteins in the SWISS‐PROT database are glycoproteins. Glycosylation plays a substantial role wide a range of physiological and pathological processes including development, immunology, cancer, and infectious disease. Protein glycosylation is also vitally important in the development of therapeutic bioproducts. Currently, more than 165 recombinant protein pharmaceuticals are approved for human use, with another 500 in preclinical and clinical trials. Of these, approximately 70% are glycosylated proteins. Glycosylation affects the structure, activity, immunogenicity, protease sensitivity, stability, and biological clearance of glycoproteins. Hence, an understanding of the mechanisms by which proteins are glycosylated, and strategies for analyzing and controlling glycoforms has become increasingly important in the development of biopharmaceuticals. Advances in chromatography and mass spectrometry have permitted more detailed identification of glycans, while cellular and protein engineering strategies have allowed manipulation of the glycoforms. In this chapter, we review the biology of protein glycosylation, methods for identifying and characterizing glycans and glycoproteins, and the effects of host cell line, culture conditions, and cellular engineering on the glycoforms of recombinant glycoproteins, providing a comprehensive overview of glycosylation of recombinant protein therapeutics.

Glycoprofiling effects of media additives on IgG produced by CHO cells in fed‐batch bioreactors

“…Since glycosylation is an important parameter for IgG function, strategies to modify glycosylation profiles of human IgG or to select the most efficient glycoforms have been explored. While genetic engineering has been employed in many cases, it is often simpler to implement changes to the cell culture medium (For a review, see Andersen et al, ).…”

“…The investigated compounds include D‐(+)‐Mannose, D‐galactose, L‐fucose, GlcNAc, ManNAc, N‐Acetylneuraminic acid (NeuNAc), uridine, and cytidine. To our knowledge none of these compounds have been investigated in an IgG‐producing CHO cell line before, and the results for especially D‐galactose and ManNAc have been highly divergent in the conditions studied (Andersen et al, ). Analysis of the effect of NeuNAc has not been performed anywhere, to our knowledge.…”

An 'omics approach towards CHO cell engineering

“…Very low concentrations of glucose in the media adversely impact cell viability and decrease glycosylation‐site occupancy in the transgenic glycoprotein product (Hayter et al, 1992). Media components such as copper sulfate, manganese sulfate, and zinc sulfate and bioreactor temperature, pH, and shear stress can also impact cell growth and viability as well as quantity and quality of the transgenic protein produced (Andersen et al, 2011; Dahodwala et al, 2012; Nam et al, 2008). Additional additives such as sodium butyrate can inactivate histone deacetylases, increasing histone acetylation, and increasing expression of the transgene (Jiang and Sharfstein, 2008).…”

“…Techniques that are currently used for analyzing glycoproteins, proteoglycans, and glycolipids and their attached glycans include high‐performance anionic exchange chromatography with pulsed amperometric detection, micellar electrokinetic capillary chromatography, capillary isoelectric focusing, capillary zone electrophoresis, matrix‐assisted laser desorption ionization mass spectrometry, capillary electrophoresis with laser‐induced fluorescence, hydrophilic interaction liquid chromatography, weak anionic exchange, reverse phase, electrospray ionization tandem mass spectrometry, matrix‐assisted laser adsorption–desorption ionization time of flight, collision‐induced dissociation, electron transfer dissociation, and electron capture dissociation. A detailed review of the techniques for analyzing protein glycosylation has recently been published (Andersen et al, 2011). Newly developed techniques for analyzing glycans may help to decipher the structure of the glycans in CHO cells.…”

“…Incorrect glycosylation can create a product that is non‐functional, has an accelerated clearance rate, or is immunogenic. Beyond simple relationships (e.g., feeding galactose increases galactosylation), there is little understanding of how process conditions affect product quality, again requiring empirical optimization (Andersen et al, 2011).…”