Department of Chemical Engineering and Materials Science, University of California at Davis, 95616, USA.
Biotechnol Bioeng. 2010 Jun 15;106(3):408-21. doi: 10.1002/bit.22713.
Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post-translational modification. In this study, bioreactor production of recombinant human alpha-1-antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25-fold increase in extracellular functional rAAT (603 microg/L) and a higher ratio of functional rAAT to total rAAT (85-90%). Surprisingly, sustained rAAT production and steady state, long-term bioreactor operation is possible following chemical induction and establishment of the viral amplicons.
植物细胞培养是生产重组人治疗性蛋白的一种替代方法，因为它可以提高产品安全性、降低生产成本，并具有真核生物翻译后修饰的能力。在这项研究中，使用化学诱导型黄瓜花叶病毒（CMV）病毒扩增子表达系统在转基因烟草细胞培养物中生产重组人α-1-抗胰蛋白酶（rAAT）糖蛋白。优化化学诱导型植物细胞培养需要评估诱导时机（TOI）和诱导剂浓度（COI）对蛋白质产率和蛋白质质量（生物功能）的影响。为了确定最佳的 TOI，选择植物细胞培养的耗氧率（OUR）作为诱导最大 rAAT 表达的生理指标。使用半连续培养法研究了 COI 对 rAAT 生产的影响，该方法可以区分生长速率和诱导剂浓度的影响。进一步提出了优化的半连续生物反应器操作，以最大限度地提高重组蛋白的产量。结果表明，与传统的分批生物反应器操作相比，转化了可诱导病毒扩增子表达系统的转基因植物细胞在优化的半连续生物反应器过程中保持更高的 OUR，并表现出较低的细胞外蛋白酶活性和较低的总酚浓度，导致细胞外功能性 rAAT（603 μg/L）增加 25 倍，功能性 rAAT 与总 rAAT 的比例（85-90%）更高。令人惊讶的是，在化学诱导和病毒扩增子建立后，可持续的 rAAT 生产和稳定、长期的生物反应器操作是可能的。
