Trentmann Oliver, Khatri Narendar K, Hoffmann Frank
Novoplant GmbH, Am Schwabeplan 1b, D-06466 Gatersleben, Germany.
Biotechnol Prog. 2004 Nov-Dec;20(6):1766-75. doi: 10.1021/bp049711h.
A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.
一种针对产肠毒素大肠杆菌菌毛的单链抗体片段由重组巴斯德毕赤酵母在甲醇诱导型AOX1启动子的控制下产生。在限定培养基上进行高细胞密度培养时，比较了甲醇限制和甲醇饱和条件。在甘油上进行分批和补料分批阶段后，将甲醇浓度控制在1%（v/v）或按指数增加速率进料甲醇。甲醇限制损害细胞完整性和产品质量，最终由于降解而未产生活性产品，而氧气限制是可以接受的。为了推迟限制的开始，通过纯氧富集进气。然而，由于甲醇消耗更快，该过程对进料速率的波动变得敏感，并且在小扰动时遇到的代谢完全停止缩短了活性生产期。在没有额外氧气供应的情况下，该过程是稳健的。通过流式细胞术监测培养物完整性的丧失，发现其先于代谢速率的变化；因此它可以作为即将出现问题的敏感指标。当省略在pH滴定后会凝结的消泡剂时，通过His-亲和色谱从培养上清液进行单步下游处理是有效的，并且从6 L初始培养体积中可得到1 g纯化的冻干产品。
