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Review
. 2022 Jun;119(6):1343-1358.
doi: 10.1002/bit.28066. Epub 2022 Feb 28.

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation

Elizabeth Edwards  1 , Maria Livanos  1 , Anja Krueger  2 , Anne Dell  2 , Stuart M Haslam  2 , C Mark Smales  3   4 , Daniel G Bracewell  1
Affiliations
Review

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation

Elizabeth Edwards et al. Biotechnol Bioeng. 2022 Jun.

Abstract

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies.

Keywords: CQAs; N-glycosylation; antibodies; biosimilars; downstream glycosylation bioprocessing; mAbs.

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Figures

Figure 1
Figure 1
Simplified N‐glycan structure scheme showing the complexity of matured biantennary N‐glycan structures, general N‐glycan nomenclature and common N‐glycan structures seen attached to Asn297 in the Fc region of IgG1. Reducing and nonreducing terminology is applied from basic glycobiology. Bisecting N‐glycans are common in human serum. Complex N‐glycans follow a complex core structure comprising five sugar residues, three mannoses, and two N‐acetylglucosamine (GlcNAc) residues. The immature N‐glycan is normally trimmed back until the core structure is created to achieve full complexity of the maturing N‐glycan required for monoclonal antibodies (mAbs). N‐glycans in general can reach higher branching
Figure 2
Figure 2
Monoclonal antibody (mAb) mechanism of action by way of antibody‐dependent cellular cytotoxicity (ADCC). The antigen binding (Fab) region of the mAb binds the antigen on the surface of a target cell, such as a cancer cell. The Fc region then binds an Fc γ‐receptor (Fc γR) on the surface of an immune effector cell, such as a natural killer (NK) cell. This cross‐linking stimulates the NK cell to release apoptotic agents that kill off the cancer cell in a targeted manner. IgG are glycosylated in the Fc region and sometimes also in the Fab region, and these glycans mediate clinically relevant properties such as serum half‐life, pharmacokinetics, target binding affinity, and overall therapeutic efficacy. Created with BioRender.com
Figure 3
Figure 3
Schematic of the monoclonal antibody (mAb) N‐glycosylation pathway that occurs during mammalian cell culture and the possible stages at which heterogeneity can arise. (1) an immature precursor N‐glycan (three glucose, nine mannose, and two N‐acetylglucosamine residues, Glc3Man9GlcNAc2) is transferred from the dolichol phosphate anchor onto an asparagine (Asn) residue on the nascent mAb protein within an Asn‐X‐Ser/Thr, except Pro motif within the primary protein structure. (2) The three terminal Glc residues are further cleaved by a set of glucose specific glucosidases. (3) The immature N‐glycan is further trimmed to an immature Man8 N‐glycan structure. (4) The immature glycoprotein is transported from the endoplasmic reticulum (ER) to the Golgi apparatus (Golgi) via vesicular trafficking. Further trimming to a Man5 structure occurs in the Golgi. (5) ββ‐1,2‐N‐acetylglucosaminyltransferase I adds a GlcNAc residue to one of the terminal mannose residues. (6) Two terminal mannose residues are trimmed by α‐mannosidase II. (7) Another N‐acetylglucosyltransferase adds a GlcNAc residue onto the terminal mannose residue, followed by a fucosyltransferase adding a fucose sugar to the core GlcNAc. (8) Two galactose (Gal) residues are added to the two terminal GlcNAcs by specific galactosyltransferases. (9) Two sialic acid residues are added to the two terminal Gals by specific sialyltransferases and, finally, (10) the glycoprotein is encased in a vesicle, which buds off from the Golgi and travels towards the cell surface. There are about 100 glycosyltransferases and glucosidases involved in the processing and trimming of N‐glycans and other glycoconjugates. Most biosynthetic steps are precursor‐dependent, which gives rise to an immense variety of glycans described as heterogeneity. Created with BioRender.com
Figure 4
Figure 4
Monoclonal antibody (mAb) glycosylation pathway in Chinese hamster ovary (CHO) cells with the FUT8 gene knocked out. This engineered pathway yields IgG mAbs with afucosylated glycans attached to Asn297. Created with BioRender.com
Figure 5
Figure 5
Monoclonal antibody (mAb) glycosylation pathway of Chinese hamster ovary (CHO) cells when diverted towards N‐acetylglucosamine (GlcNAc) bisection. The overexpression of β1,4‐N‐acetylglucosaminyltransferase III (GnTIII) generates bisected glycans, which cannot be fucosylated due to the steric hindrance created by the GlcNAc residue for c1,6‐FucT. Created with BioRender.com
Figure 6
Figure 6
Schematic of the fucosylated and afucosylated IgG glycoform separation afforded by glycosylated FcγRIIIa‐based ligands. The pool of IgG loaded onto the column is heterogeneously glycosylated and a gradient elution (red line) is used. Fucosylated IgG binds the ligands with lower affinity and therefore elutes off the column first. As the elution gradient increases, the more tightly bound afucosylated IgG eventually also elutes. Created with BioRender.com
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