Production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures in a pilot‐scale bioreactor

Abstract

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low‐cost production, although low yields remain the major challenge. The studies on scaling‐up to pilot‐scale bioreactors, especially in conventional stainless‐steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled‐up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40‐L pilot‐scale STB. Employing cyclical operation together with a simplified‐process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab‐scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46–58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot‐scale STB to be equivalent to the lab‐scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L−1 day−1, respectively, which are comparable to the lab‐scale culture. Here, we demonstrate pilot‐scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long‐term, reproducible, and sustained production of rrBChE.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

“…The initial transgenic Z4 line callus was maintained and propagated in MYT solid medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/L), 6-benzylaminopurine (BA, 0.3 mg/L), adenine (10 mg/L), and a 20 mixture amino acids cocktail [20]. However, the influence of the cultivation strategy on the cost of recombinant protein production is essential to identify cost-effective bioreactor operation conditions [21]. We found a suitable cell growth using liquid MS basal medium supplemented with 2,4-D (2 mg/L) and kinetin (2 mg/L) for carrot cell suspension.…”

Immobilization of transgenic plant cells towards bioprinting for production of a recombinant biodefense agent

“…The ability to immobilize, and eventually bioprint, plant cells for use in a continuous flow‐through bioreactor configuration offers a production platform for numerous therapeutics with the potential to overcome the limitations associated with current plant cell culture processes. Typically, plant cells are cultured in suspension in stirred, air‐sparged bioreactors [ 4,47–50 ] or in a “packed bed” of plant cells using a membrane bioreactor, [ 51 ] and in these conditions have been shown to maintain viability and productivity for several months. However, neither of these bioreactor systems allow precise control over the 3D position of the plant cell aggregates, cell aggregate size distribution, or nutrient and product concentration profiles, which can affect productivity, ease of product recovery, and product quality.…”

Bioprinting transgenic plant cells for production of a recombinant biodefense agent

“…The ability to 3D-print a plant cell bioink for use in a continuous flow-through bioreactor configuration offers a production platform for numerous therapeutics with the potential to overcome the limitations associated with a diversity of current plant cell culture processes. Typically, plant cells are cultured in suspension in stirred, air-sparged bioreactors [4,43–46] or in a “packed bed” of plant cells using a membrane bioreactor [47], and in these conditions have been shown to maintain viability and productivity for several months. However, neither of these bioreactor systems allow precise control over the 3D position of the plant cell aggregates, cell aggregate size distribution, or nutrient and product concentration profiles, which can affect productivity, ease of product recovery, and product quality.…”

“…The ability to 3D-print a plant cell bioink for use in a continuous flow-through bioreactor configuration offers a production platform for numerous therapeutics with the potential to overcome the limitations associated with a diversity of current plant cell culture processes. Typically, plant cells are cultured in suspension in stirred, air-sparged bioreactors [4,[43][44][45][46] or in a "packed bed" of plant cells using a membrane bioreactor [47], and in these conditions have been shown to maintain viability and productivity for several months.…”