Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture

Abstract

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10-20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8 mg L(-1) over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N-glycan analysis.

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…In situ protein recovery from the medium -Yes foam and interfere with downstream processing, have been identified as significant practical problems associated with exogenous stabilising agents in plant culture media [78,86].…”

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

“…We used the plant expression vector pTRAkc-2G12-DsRed [20], containing the genes for the 2G12 antibody heavy chain and light chain as well as the gene for DsRed, each under the control of a cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer [21], on a single T-DNA. The three expression cassettes were separated by scaffold attachment regions and the vector was introduced into A. tumefaciens strain GV3101:pMP90RK [11].…”

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

“…In fact, it can affect the solubility, stability and biological activity of a protein positively as well as negatively [17]. In contrast to mammalian cell-based production where a mixture of N -glycans is often present on recombinant proteins, the N -glycans produced in plants are very homogenous within/along a given protein molecule as well as between batches [16,134,138]. This opens opportunities for N -glycan-dependent therapeutics, for example, those for treatment of lysosomal storage diseases.…”

“…The commercial success by these two companies undoubtedly ushers in a new era in the biopharmaceutical industry that promises to provide growth opportunity for this new platform. Other plant cell-made pharmaceuticals, including monoclonal antibodies [14–16], vaccines [17–20], growth factors [21] and cytokines [22–25] that are in pre- and early clinical stages, as well as more therapeutic enzymes in the Protalix’s development pipeline [11], are expected to enter into the marketplace in the future. Plant cell culture is now reaching the stage at which it may challenge those established bioproduction systems that use bacterial, yeast and mammalian cells, though major problems with plant cell culture still exist with regards to low product yields, inherent production variability and nonmammalian glycosylation.…”