Su-Hwan Cheon

Bioreactor engineering using disposable technology for enhanced production of hCTLA4Ig in transgenic rice cell cultures

Two kinds of disposable bioreactors, air‐lift disposable bioreactors (ADB) and wave disposable bioreactors (WDB) were compared with stirred‐tank reactors (5‐L STR). These bioreactors were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T‐lymphocyte antigen 4‐immunoglobulin (hCTLA4Ig). In both systems, a fed‐batch culture method was used to produce hCTLA4Ig efficiently by feeding concentrated amino acids and production levels were enhanced when dissolved oxygen (DO) level was regulated at 30% using pure oxygen sparging. Agitation and aeration rate during cultivation in ADB and WDB were determined by the same mixing time. The results in both disposable bioreactors showed similar values in maximum cell density (11.9 gDCW/L and 12.6 gDCW/L), doubling time (4.8‐ and 5.0‐day), and maximum hCTLA4Ig concentration (43.7 and 43.3 mg/L). Relatively higher cell viability was sustained in the ADB whereas hCTLA4Ig productivity was 1.2‐fold higher than that in WDB. The productivity was improved by increasing aeration rate (0.2 vvm). Overall, our experiments demonstrate pneumatically driven disposable bioreactors are applicable for the production of recombinant proteins in plant cell cultures. These results will be useful for development and scale‐up studies of disposable bioreactor systems for transgenic plant cell cultures. Biotechnol. Bioeng. 2013; 110:2412–2424.

Production of biopharmaceuticals in transgenic plant cell suspension cultures

Abstract Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effec-tiveness, scalability and possibility of complex post-trans-lational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

Application of anoxia with glucose addition for the enhanced production of hCTLA4Ig in transgenic rice suspension cell cultures

To enhance the production of hCTLA4Ig in transgenic rice suspension cell cultures, anoxic conditions were applied during the production phase. Under the anoxic conditions in sugar-depleted media, cell viability was reduced rapidly and protease activity increased compared to aerobic conditions. However, the maximum production level of hCTLA4Ig with sugar-depleted anoxic conditions was the same as that in aerobic conditions. In addition, the production of hCTLA4Ig under anoxic conditions reached a peak 2 days earlier than that in aerobic conditions. Addition of 30 mM glucose at the production phase under anoxic conditions markedly improved cell viability. A viability level over 65% could be maintained for more than 30 days. Repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxic conditions with 30 mM glucose. In addition, the production periods of hCTLA4Ig was extended up to 30 days and the maximum production level of hCTLA4Ig under anoxic conditions was 2.1-fold higher. Therefore, anoxic conditions could be used for the enhanced production of hCTLA4Ig in transgenic rice cell cultures.