Abstract
The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)−1 or 7.5 ± 0.4 mg L−1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.
Highlights
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
The reduction of biomass during sugar starvation was noticed in our previous reports [39,43], suggesting that kifunensine may not adversely affect rice cell mass cultured in 1.25X NB-S, even though a negative impact on rice cell mass cultured in Chu medium
Performing a media exchange using 1.25-times-concentrated sugar-free medium (NB-S) together with 1.25-times-reduced culture volume and addition of kifunensine prior to and after the media exchange resulted in increased total production levels of active rice recombinant human BChE (rrBChE), volumetric productivity, and specific productivity by 1.5 times, 3.4 times, and 1.5 times, respectively, compared with a bioreactor run with same operating conditions (27 ◦C, 75 rpm, 0.2 vvm) but no kifunensine treatment
Summary
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]. Of particular concern are plant-specific structures contained in complex type N-glycans, namely, α1,3 core fucose, β1,2 bisecting xylose, and the Lewis a epitope [8]. Even though there is no definitive proof of adverse effects from plant-specific glycan structures [9], the presence of nonhuman glycans could potentially cause unwanted immunogenicity in humans, and the lack of sialic acid termination may lead to reduced blood circulatory half-life. 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
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