Artificial Golgi reactions for targeted glycosylation

Abstract

N-linked glycosylation constitutes a Critical Quality Attribute for biotherapeutics. It is known to affect drug efficiency, efficacy and half-life. Glycosylation is a non-templated and complex process owning firstly to the promiscuity of the enzymes involved and secondly to enzyme and nucleotide sugar donor availability. This leads to heterogeneity amongst cell-derived glycoproteins, limiting therapeutic efficacy. Production of biotherapeutics focuses on controlling the glycosylation profile to enhance their activity and produce tailored drugs. Despite the intense efforts to control glycosylation, current methods face important limitations including simplicity, cost and lack of homogeneity. The work presented here addresses the current limitations by developing an Artificial Golgi Reactor (AGR) that allows bespoke N-linked glycosylation of glycoproteins in an artificial environment. Specifically, this novel proof-of-concept system comprises immobilised glycosyltransferase (GnTI, GalT) and glycosidase enzymes (ManII). These enzymes comprise a glycosylation pathway where promiscuity naturally exists. A method to express, in vivo biotinylate and immobilise GnTI and GalT was developed enabling “one-step immobilisation/purification”. ManII was biotinylated using an alternative chemical approach and similarly immobilised. The immobilised enzymes were used in a sequential fashion to reconstruct the N-linked glycosylation pathway on artificial glycans and on a monomeric Fc expressed in glycoengineered Pichia pastoris. The spatiotemporal separation tackled enzyme promiscuity, resulting in increased glycoform homogeneity (>95% conversion). Finally, immobilised GalT was used to enhance the galactosylation profile of three IgGs, yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140-hours. The methods and results outlined in this work demonstrate the application of the AGR as an in vitro glycosylation strategy applied post-expression that is easy to implement, modular and reusable. Furthermore, it has the potential to be expanded and applied for the large-scale manufacture of bespoke biotherapeutics.