Glycoengineering of Therapeutic Glycoproteins:  In Vitro Galactosylation and Sialylation of Glycoproteins with Terminal N-Acetylglucosamine and Galactose Residues

Abstract

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.

Production of Highly Sialylated Monoclonal Antibodies

“…Several approaches are used to enhance the sialylation level of recombinant proteins produced in mammalian cell cultures, such as cell line engineering, bioprocess control, postproduction enrichment (such as lectin-affinity purification) and in vitro glycan remodelling [27,40]. In the field of mammalian cell engineering, a variety of approaches were developed.…”

Production of α2,6-sialylated IgG1 in CHO cells

“…The in vitro enzymatic addition of galactose and sialic acid residues on purified mAbs was shown to be effective, but requires several 24 hour-cycles of reaction, substantial amounts of costly recombinant enzymes and substrates, and an additional purification step. 35,38,39 Single amino acid mutations in the Fc, such as the replacement of the phenylalanine 243 by an alanine that disrupts the glycan-CH2 domain interaction, 33,40 were shown to allow enhanced galactosylation and sialylation, reinforcing the hypothesis that glycan embedment and interaction with the Fc structure may limit its access to some glycosyltransferases. However, amino acid substitutions lying far away from the glycan were also shown to affect glycosylation and receptor binding.…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…Sialic acid terminating glycan moieties can also strongly modulate the efficacy of mAbs. Interestingly, only a small proportion of human IgG is naturally sialylated [38], which can be directly related to the spatial constraints in the C 2 domain of IgG antibodies [39]. Sialic acid can be found in either a 2,3-or 2,6-linkage to the galactose moieties of IgGs [40], and although not very abundant, these residues are crucial for immune response modulation (anti-inflammatory activity) in vivo, where the 2,6-linkage is preferred over the 2,3 [41].…”