Bioprocess Clinical Manufacturing and Technology, Merck Research Laboratories, Merck & Co., Inc., PO Box 4, West Point, PA 19486, USA.
Biotechnol Prog. 2011 May-Jun;27(3):657-67. doi: 10.1002/btpr.560. Epub 2011 Apr 11.
Large-scale fermentation of Pichia pastoris requires a large volume of methanol feed during the induction phase. However, a large volume of methanol feed is difficult to use in the processing suite because of the inconvenience of constant monitoring, manual manipulation steps, and fire and explosion hazards. To optimize and improve safety of the methanol feed process, a novel automated methanol feed system has been designed and implemented for industrial fermentation of P. pastoris. Details of the design of the methanol feed system are described. The main goals of the design were to automate the methanol feed process and to minimize the hazardous risks associated with storing and handling large quantities of methanol in the processing area. The methanol feed system is composed of two main components: a bulk feed (BF) system and up to three portable process feed (PF) systems. The BF system automatically delivers methanol from a central location to the portable PF system. The PF system provides precise flow control of linear, step, or exponential feed of methanol to the fermenter. Pilot-scale fermentations with linear and exponential methanol feeds were conducted using two Mut(+) (methanol utilization plus) strains, one expressing a recombinant therapeutic protein and the other a monoclonal antibody. Results show that the methanol feed system is accurate, safe, and efficient. The feed rates for both linear and exponential feed methods were within ± 5% of the set points, and the total amount of methanol fed was within 1% of the targeted volume.
毕赤酵母的大规模发酵在诱导阶段需要大量甲醇进料。然而，由于持续监控、手动操作步骤以及火灾和爆炸危险等方面的不便，大量甲醇进料很难在加工车间中使用。为了优化和提高甲醇进料过程的安全性，设计并实施了一种新颖的自动化甲醇进料系统，用于毕赤酵母的工业发酵。描述了甲醇进料系统的设计细节。设计的主要目标是实现甲醇进料过程的自动化，并最大限度地降低在加工区域储存和处理大量甲醇所带来的危险风险。甲醇进料系统由两个主要组件组成：大容量进料（BF）系统和多达三个便携式过程进料（PF）系统。BF 系统从中央位置自动将甲醇输送到便携式 PF 系统。PF 系统为发酵罐提供线性、阶梯或指数甲醇进料的精确流量控制。使用两种 Mut(+)（甲醇利用增强）菌株进行了线性和指数甲醇进料的中试发酵，一种表达重组治疗蛋白，另一种表达单克隆抗体。结果表明，甲醇进料系统准确、安全且高效。线性和指数进料方法的进料速率均在设定点的±5%范围内，并且所进料的甲醇总量与目标体积相差 1%以内。
