Abstract
N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.
Highlights
Therapeutic glycoproteins represent a predominant disease treatment category among biopharmaceuticals approved or in clinical development
All non-engineered eukaryotic cells share a common N-glycosylation core, while terminal and lateral oligosaccharide residues are specific to each expression system
Design of a manufacturing process allowing for precise regulation of protein N-glycosylation is highly advantageous and urgently needed, as N-glycosylation often profoundly affects biological efficacy and pharmacokinetic properties of the manufactured drug substance [4,5,6,7]
Summary
Therapeutic glycoproteins represent a predominant disease treatment category among biopharmaceuticals approved or in clinical development. Several manufacturing approaches have been considered to modify the host N-glycosylation machinery, with the goal of exploiting the benefit of specific glycosylation profiles and generating therapeutics with targeted biological functions These included modulation of cell growth conditions [2], silencing or overexpression of glycan processing enzymes [20,21,22], complete or partial removal of glycans [23,24,25], and in vitro glycosylation remodeling [26,27]. Knock-in strategies have been employed to introduce or modify the sialylation pathway and provide recombinant proteins with human-type sialylation; offering higher circulatory half-life and reduced product immunogenicity [35,36,37] This type of approach generally enhanced human-type protein sialylation, and increased glycoform heterogeneity. 3 of 17 3 of 17 developed for large-scale therapeutics manufacturing in the well-characterized Nicotiana benthamiana pmlaannt usfyasctteumri.nIgnicnortphoerwateinllg-chparoratecitnerigzleydcaNnicmotoiaientaybreenmthoamdeialinnagpilnatnot sthyisstepmla.nItnceoxrpproersasitoinngspyrsotetemin dgelmycoannstmraotieestythreemprooddeulcintigoninotof athsiisnpglleangtlyecxopfroersmsiornitusyxsimteamb dheamrboonrisntrgatheusmthaenp-lrikoeduNc-tgiolyncoofsyalasitniognle agnldycnoofoprlamntr-istpuexciimficabsuhgaarrbroersiindguehsu. mThains-nliokveeNl a-pgplyrcooascyhlastimionpliafnieds annoepxlpaennt-ssipveecgilfiyccasnugreamr roedseidliunegs. pTrohcisednuovreelfaacpiplirtaotaicnhgsaimcopslti-fieeffseacntiveexpaenndsisvcealgalbylceapnrroecmesosd. eling procedure facilitating a cost-effective 2a. nRdessucaltlsable process
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