title: Pneumatic hydrodynamics influence transplastomic protein yields and biological responses duringin vitroshoot regeneration ofNicotiana tabacumcallus: Implications for bioprocess routes to plant-made biopharmaceuticals.
abstract: Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomicNicotiana tabacumshoots from callus tissue in RITA®temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min-1was identified, corresponding to a shear rate of 96.7 s-1, pneumatic power input of 8.8 mW kg-1and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.
authors.affiliation: Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Sherwin S
authors.initials: SS
authors.lastname: Barretto
authors.affiliation: Alkion Biopharma SAS, Pépinière Entreprise Genopole, 4 rue Pierre Fontaine, 91058, Evry, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Michoux
authors.affiliation: Department of Chemical Engineering, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Klaus
authors.initials: K
authors.lastname: Hellgardt
authors.affiliation: Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nixon
keywords: Biopharmaceutical; CIM, callus induction medium; Hydrodynamics; MS medium, Murashige & Skoog medium; Pneumatic energy dissipation; RITA®, recipient for automated temporary immersion (translated from French); SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TF, triphenylformazan; TIB, temporary immersion bioreactor; TSP, total soluble protein; TTC, 2,3,5-triphenyltetrazolium chloride; Temporary immersion culture; TetC, fragment C of tetanus toxin; Transplastomic protein; in vitro organogenesis; kDa, kiloDalton