On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect

Abstract

Plant cell culture is emerging as an alternative bioproduction system for recombinant pharmaceuticals. Growing plant cells in vitro under controlled environmental conditions allows for precise control over cell growth and protein production, batch-to-batch product consistency and a production process aligned with current good manufacturing practices. With the recent US FDA approval and commercialization of the world's first plant cell-based recombinant pharmaceutical for human use, β-glucocerebrosidase for treatment of Gaucher's disease, a new era has come in which plant cell culture shows high potential to displace some established platform technologies in niche markets. This review updates the progress in plant cell culture processing technology, highlights recent commercial successes and discusses the challenges that must be overcome to make this platform commercially viable.

Figure 1. Suspension cultured tobacco BY-2 cells for recombinant protein expression

(A) BY-2 cells grown in a shake flask for a week; (B) Fluorescence micrographs of BY2 cells expressing enhanced green fluorescence protein. The cells were inspected for green fluorescence using a Zeiss LSM 510 laser-scanning confocal microscope (Carl Zeiss AG, Jena, Germany) (488 nm excitation; 510 nm emission). The cultured plant cells are clustered, and most of the expressed enhanced green fluorescence protein protein is accumulated at the cell wall-cytoplasm membrane interface.

Figure 2. Recombinant human β-glucocerebrosidase production with carrot cell culture by Protalix BioTherapeutics

(A) GCD expression cassette constructed in the binary vector pGREENII. The expression cassette comprises the CaMV35S promoter, the TMV omega translational enhancer, a signal peptide, the human GCD sequence, a vacuolar targeting signal and the octopine synthase terminator sequence from Agrobacterium tumefaciens [46]. (B) Carrot cell suspension culture in disposable plastic bioreactors for the production of GCD [11]. (C) Two major N-linked glycan structures detected on the recombinant GCD expressed in carrot cells. These N-glycans have a main core of two N-acetylglucosamine residues and a β1–4-linked Mannose, attached to two additional mannose residues in α1–3 and α1–6 linkages (shadowed) [46]. GCD: β-glucocerebrosidase; TMV: Tobacco mosaic virus.

Figure 3. Enhanced secreted protein yields by the HypGlyco technology in plant cell culture

(A) Schematic of HypGlyco technology. Here, all the ‘Pro’ residues in the ‘Ser–Pro’ module, or (SP) for short, are hydroxylated to be Hyp and subsequently O-glycosylated with arabinogalactan polysaccharides in plant cells; (B) Enhanced secretion of EGFP by an N-terminal HypGlyco carrier (SP)₃₂ in tobacco BY-2 cell culture. The (SP)₃₂ refers to 32 tandem repeats of the ‘Ser–Pro’ dipeptide motif, which dramatically enhanced the secretion of the tagged EGFP from the culture tobacco cells with more than 250 mg/l of secreted EGFP detected. By comparison, EGFP expressed without a HypGlyco carrier was barely detectable in the culture medium (<1.0 mg/l); (C) SDS-PAGE separation of the culture media of the tobacco cells expressing (SP)₃₂–EGFP. The media were harvested every other day for 14 days. The Coomassie blue-stained SDS-PAGE gel showed the (SP)₃₂–EGFP fusion protein dominated the cell culture media; (D) Reversed-phase HPLC detection of the dominant (SP)₃₂–EGFP peak in the cell culture medium (after 12 days’ culture). d: Days; EGFP: Enhanced green fluorescence protein.