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

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.

Effects of Kifunensine on Production and <em>N</em>-glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

“…The reason for the N-glycoform shift from predominantly 3211 to 4601 at site N241 in ME(+) ( Figure S1) is unclear; however, it does not imply that rrBChE gains or loses activity and/or interaction with the tetramerization domain since the N241 site remains highly glycosylated. At site N341, the abundance of the In plant-based systems, several studies have reported that glycoproteins treated with kifunensine contain predominantly Man9 structures [4,23,24] and Man7/8/9 structures [25], while Man5/6/7 was the main structure found in this study. The discrepancy between our finding and others is likely due to the mass transfer limitation of kifunensine.…”

“…Protein expression in plant systems has the potential to provide a safe, cost-effective, and scalable method to meet the increasing need for therapeutic protein production. Plantbased expression offers several advantages to the biopharmaceutical industry, including decreased cost of production, scalability, a lack of susceptibility to mammalian pathogens, elimination of animal or human sourced raw materials, and the ability to produce complex proteins with post-translational modifications such as N-glycosylation [1][2][3][4][5]. For many therapeutic proteins, N-glycosylation is essential for protein folding, oligomerization, quality control, enzyme activity, ligand interactions, localization, and trafficking [6,7].…”

“…Therefore, to ensure the efficacy of a plant-made biosimilar therapeutic, it is important that the N-glycans are compatible with both the protein's function and the human immune system. There are several strategies for modifying a glycoprotein's N-glycan structures in planta, such as glycoengineering of the host cells using CRISPR/Cas9 genome editing to knock out β(1,2)-xylosyltransferase (XylT) genes and α(1,3)-fucosyltransferase (FucT) genes [10][11][12] and RNA interference (RNAi) technology to down regulate XylT and FucT genes [13][14][15][16][17], targeting of the protein to specific organelles [18][19][20][21], addition of compounds to alter the function of glycan-modifying enzymes [4,[22][23][24][25][26], and in vitro glycan remodeling using chemoenzymatic reaction [27]. In this work, we utilize the last strategy by adding kifunensine, a potent and highly specific inhibitor of mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 (Man8) and Man9GlcNAc2 (Man9) structures [28], to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity.…”

“…In this work, we utilize the last strategy by adding kifunensine, a potent and highly specific inhibitor of mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 (Man8) and Man9GlcNAc2 (Man9) structures [28], to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity. More than a few studies of kifunensine treatment in whole Nicotiana benthamiana plants successfully produced predominant Man9 structure glycoproteins [4,[22][23][24]26]; however, the study of kifunensine treatment in plant cell suspension cultures, including transgenic rice cell suspensions [25], is very limited. In transgenic rice cell suspensions treated with 5 μM kifunensine cultivated in shake flasks, the productivity of a target glycoprotein, acid α-glucosidase (GAA), was significantly lower than the control, but the relative abundance of high mannose structure GAA increased 65% compared to the control [25].…”

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

“…Protein expression in plant systems has the potential to provide a safe, cost-effective, and scalable method to meet the increasing need for therapeutic protein production. Plant-based expression offers several advantages to the biopharmaceutical industry, including decreased cost of production, scalability, lack of susceptibility to mammalian pathogens, elimination of animal- or human-sourced raw materials, and the production of complex proteins with post-translational modifications such as N -glycosylation [ 1 , 2 , 3 , 4 , 5 ]. For many therapeutic proteins, N -glycosylation is essential for protein folding, oligomerization, quality control, enzyme activity, ligand interactions, localization, and trafficking [ 6 , 7 ].…”