Process Development for the Production of Biopharmaceuticals Using Transgenic Plant Cells in Bioreactor

초록

Transgenic plant cell cultures have been evaluated as alternative platforms for the production of biopharmaceuticals due to their inherent safety and cost-effectiveness during manufacturing and purification steps. Most of the technical know-how and experience of bioreactor engineering is applicable to plant cell cultures. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a fusion recombinant protein, was expressed in transgenic rice cell suspension cultures. Expression of hCTLA4Ig was performed under the control of RAmy3D promoter. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4Ig into the media. The rice-derived hCTLA4Ig could be purified with affinity chromatography using protein A resin. hCTLA4Ig was produced in stirred tank bioreactors with various process strategies. Marine, turbine, and hollowed paddle impeller were compared for the cultivation of transgenic rice cells and optimization of culture medium was performed. Disposable technology was also applied for the cultivation of rice cells. Two-stage cultivation method was applied to achieve high cell density during cell growth phase and to enhance the production level. Two kinds of media exchange methods (perfusion and direct exchange) were compared in a 5-L stirred tank bioreactor. In the production phase, inducible production of hCTLA4Ig caused cell death because of sugar depletion, and media exchange induced cell lysis by additional shear stress. It was essential to reduce shear stress for increasing productivity because protease activity was increased by cell lysis. High density cultivation was also investigated in the bioreactor. With fed-batch cultivation, maximum dry cell weight reached 21.2 g/L and maximum hCTLA4Ig production reached 13.4 mg/L which were 2.18-fold and 2.14-fold higher than those of the direct media exchange, respectively.