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.