| Plant cell suspension culture has been used recently as an expression system for recombinant protein and antibody production, and can be advantageous compared to other systems such as microbes, yeast, mammalian culture, insect culture, animals, and whole plants. In transgenic rice cultures, a rice α-amylase (RAmy3D) inducible promoter was metabolically regulated to produce a recombinant human therapeutic protein, α1-antitrypsin (rAAT). Under sugar deprivation conditions, the RAmy3D promoter was induced for expression of rAAT, and a rice α-amylase signal peptide directed secretion of the rAAT into the culture medium. The rice cultures were scaled-up from shake flasks to a 5-L bioreactor, and the cells were induced by retaining the cells in the bioreactor and performing a single medium exchange with sugar-free medium. Secretion of the target protein permitted implementation of a cyclical, semi-continuous operation where cells were reused for consecutive growth and expression phases.; Two bioreactor runs were performed and each consisted of three reproducible cycles over a 28 day period. The viable biomass concentration and the oxygen uptake reached maximums during the growth phases, while the culture pH and the functional rAAT concentration reached maximums during the expression phases. Sustained production of functional rAAT was achieved over three cycles where maximum extracellular concentrations reached ranged from 30 to 110 mg/L. The overall volumetric productivities for the two runs were 7.7 and 4.5 mg active rAAT/(L day), respectively. A mathematical model was developed to characterize the growth and expression phases of the semi-continuous process. Kinetic parameters were obtained, and the experimental data were used to evaluate the effectiveness of the model.; Variant forms of the rAAT expressed in the transgenic rice cultures have been detected during induction, although no differences were observed in the extracellular and intracellular portions. Rice-derived rAAT consisted of seven to eight heterogeneous protein molecules possibly due to differences in glycosylation, translation variations, or terminal cleavage. The influence of inhibitors on preventing degradation of rAAT was evaluated. Glycosylation of rice-derived rAAT was confirmed using carbohydrate detection blots and deglycosylation enzymes. |
