Glycoform Modification of Secreted Recombinant Glycoproteins through Kifunensine Addition during Transient Vacuum Agroinfiltration.

Yongao Xiong,Qiongyu Li,Karen Mcdonald,Somen Nandi,Carlito Lebrilla,Muchena Kailemia

doi:10.3390/ijms19030890

Yongao Xiong, Qiongyu Li + Show 4 more

Open Access

https://doi.org/10.3390/ijms19030890

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Abstract

Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.

Highlights

Plant-based pharmaceutical production is appealing given its inexpensive facility and production cost, linear scale-up, the absence of animal pathogens, and capability to produce complex proteins and perform post-translational modifications, which overcomes one or more drawbacks of traditional recombinant protein expression systems such as animal cell culture and bacterial fermentation [1,2].Much work has been carried out using stably transformed plants, but the significantly reduced development and production timeline makes transient expression of proteins in whole plants a attractive option, cutting the time to bring critical medications to the market during a pandemic [2]
A secondary anti-Fc polyclonal antibody linked to a horseradish peroxidase (HRP) enzyme binds to the CMG2-Fc allowing colorimetric detection
Twenty wild-type 5–6-week old N. benthamiana plants were divided into experimental and control groups, agro-infiltrated and incubated for 6 days, whole leaves were extracted under identical conditions to determine protein expression

Summary

Introduction

Plant-based pharmaceutical production is appealing given its inexpensive facility and production cost, linear scale-up, the absence of animal pathogens, and capability to produce complex proteins and perform post-translational modifications, which overcomes one or more drawbacks of traditional recombinant protein expression systems such as animal cell culture and bacterial fermentation [1,2].Much work has been carried out using stably transformed plants, but the significantly reduced development and production timeline makes transient expression of proteins in whole plants a attractive option, cutting the time to bring critical medications to the market during a pandemic [2]. Late N-glycosylation maturation in the Golgi apparatus is kingdom-specific, and results in different N-glycosylation on proteins produced in plants compared with human. These plant-specific glycans may lead to potential safety issues such as hypersensitivity or allergy, as plant-specific α(1,3)-fucose and β(1,2)-xylose are known to be important IgE binding determinants of plant allergens [4]. If these plant-specific glycans are present in an injected pharmaceutical product, the glycoprotein could trigger immunological response, or at least, result in a short circulation half-life. N-glycans of proteins produced from mammals are often terminated in β(1,4)-galactose and sialic acid; sialic acid is important as it typically increases the circulation half-life of proteins [5]

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