Pharmacological significance of glycosylation in therapeutic proteins
Introduction
Therapeutic proteins currently represent the main drivers for revenue growth in the biopharmaceutical industry and have a demonstrated track record of providing safe and efficacious clinical benefit. However, while therapeutic proteins have revolutionized the treatment of many previously unmet medical needs, low activity, rapid clearance, and immunogenicity remain as significant challenges to the development of protein biopharmaceuticals. More than two-thirds of marketed therapeutic proteins, including monoclonal antibodies, are glycoproteins. Glycosylation, the most common form of post-translational modification, is the diverse, enzyme-mediated process by which oligosaccharide side chains are covalently attached to either the side chain of asparagine (N-linked) or serine/threonine (O-linked), with former being the most prominent. The oligosaccharide moiety of proteins is often essential for recognition, signaling, and interaction events within and between cells and proteins, and can play an important role in folding and defining the conformation of the protein [1]. Thus, while the biological function of most therapeutic proteins is typically defined principally by its templated amino acid component, the carbohydrate component of a therapeutic glycoprotein is often key in determining its pharmacological properties including stability, solubility/bioavailability, in vivo activity, pharmacokinetics, and immunogenicity.
For the production of recombinant therapeutic glycoproteins, mammalian cell expression systems (including CHO, murine myeloma, and HEK cell lines) have been the preferred expression host because their protein glycosylation machinery largely resembles that in humans, though certain nonhuman glycoforms can and do occur in marketed products [2••]. Glycosylation patterns of therapeutic proteins produced in mammalian expression systems are generally heterogeneous and can vary from batch to batch. The acceptable variability is thus typically defined by preclinical and clinical studies from which the roles of glycosylation in pharmacokinetics, bioavailability, clearance, and potency are established.
Industry leaders in the therapeutic glycoprotein space have explored using engineered glycosylation sites to modify the pharmacological properties of new or licensed products [3]. An alternative strategy for glycoengineering, under development particularly in the therapeutic antibody field, involves modifying glycosylation patterns of therapeutic proteins by optimizing culture conditions [4, 5], introducing enzymatic modifications, and/or by genetically engineering the mammalian expression host for enrichment or elimination of particular glycoforms [6, 7].
Recent advances in the use of highly engineered yeast and other alternative eukaryotic organisms for the production of human therapeutic glycoproteins have opened the potential for faster and lower cost development and commercialization of biologic drugs. Fully functional expression of both human IgG and rat erythropoietin (EPO) have been demonstrated in a glycoengineered Pichia pastoris yeast system [8••, 9••]. Advantages of therapeutic glycoprotein production in a glycoengineered microbial host include ease of handling and genetic manipulation, decreased cost and reduced gene-to-product cycle time, and readily available industrial scale-up of processes [9••, 10, 11, 12, 13••, 14••, 15, 16, 17, 18, 19]. A further advantage of these highly engineered glycoprotein expression systems is the potential to generate highly uniform N-linked glycosylation patterns and to fully explore the pharmacologic role of the carbohydrate fraction of a therapeutic glycoprotein.
This review provides a brief summary of the process by which glycosylation proceeds in humans, followed by examples from the literature where glycosylation is known to play a significant role in the in vivo activity or other clinically relevant pharmacology of therapeutic glycoproteins.
Section snippets
The processes of N-linked and O-linked glycosylation
N-Linked glycosylation is a post-translational modification that is conserved across yeast and other eukaryotes. N-Glycosylation begins in the endoplasmic reticulum (ER) with the transfer of a preassembled dolichol phosphate-linked 14 sugar oligosaccharide to targeted asparagine residues in a protein [19, 20]. The preferred site for N-glycosylation contains the three amino acid sequence, Asn-Xaa-Ser/Thr where the second position can be any amino acid except Pro. This oligosaccharide contains
Biological activity
Glycans often play an important role in establishing or maintaining the integrity of glycoproteins. They can enhance thermal stability, provide protection from proteolysis, improve solubility, and inhibit aggregation of proteins. Given this important function, it is to be expected that glycosylation can be involved in modulating protein–protein interactions. This is highly relevant in developing and optimizing therapeutic glycoproteins, where an understanding of the relationship of the
Conclusions
The choice of an expression host (i.e. mammalian, plant, yeast, and transgenic animals) and cell culture conditions has a significant impact on glycosylation identity and heterogeneity in recombinant glycoproteins. In turn, the glycosylation profiles can influence biological activity and clinical efficacy. It is thus of high value and importance to accurately identify glycan components and quantification of various forms. Thobhani et al. [65•] perfomed an inter-laboratory study on the
References and recommended reading
Papers of particular interest have been highlighted as:
• of special interest
•• of outstanding interest
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