Research Center of Bioprocess Engineering and Analytical Techniques, HAW - Hamburg University of Applied Sciences, Hamburg, Germany.
Biotechnol J. 2011 Apr;6(4):437-51. doi: 10.1002/biot.201000385.
The successful development of optimal multistage production processes for recombinant products with Pichia pastoris needs to meet three pre-conditions. These pre-conditions are (i) strategies for performing fully automated and observable processes, (ii) characterization of the host cell-specific reaction parameters in order to make an adapted process layout for feeding and aeration strategies, and (iii) knowledge of optimal operation parameter conditions for maximizing the expression productivity of target protein amount and/or quality. In this report, an approach of a fully automated multi-bioreactor plant is described that meets all these requirements. The expression and secretion of a potential malaria vaccine with Pichia pastoris was chosen as an example to demonstrate the quality of the bioreactor system. Methods for the simultaneous identification of reaction kinetics were developed for strain characterization. Process optimization was carried out by applying a sequential/parallel Design of Experiments. In the view of Process Analytical Technology (PAT)-applications and in order to develop fully automated and globally observable production processes, methods for quasi on-line monitoring of recombinant protein secretion titers and the immunological quality of the products are also discussed in detail.
毕赤酵母表达重组蛋白的最优多阶段生产工艺的成功开发需要满足三个前提条件。这些前提条件是：（i）制定完全自动化和可观测的工艺策略，（ii）对宿主细胞特异性反应参数进行表征，以便为补料和通气策略制定适应性的工艺布局，以及（iii）了解最佳操作参数条件，以最大化目标蛋白量和/或质量的表达生产力。在本报告中，描述了一种满足所有这些要求的全自动多生物反应器工厂的方法。选择毕赤酵母表达和分泌一种潜在的疟疾疫苗作为示例，以证明生物反应器系统的质量。针对菌株表征开发了用于同时鉴定反应动力学的方法。通过应用序贯/并行实验设计进行了工艺优化。为了满足过程分析技术（PAT）应用的要求，并开发完全自动化和全局可观测的生产工艺，还详细讨论了用于准在线监测重组蛋白分泌滴度和产品免疫质量的方法。
