Methanol induction optimization for scFv antibody fragment production in Pichia pastoris
Methanol induction optimization for scFv antibody fragment production in Pichia pastoris
复制标题
DOI：
10.1002/bit.20051
复制
发表时间：
2004-05-20
影响因子：
3.8
通讯作者：
Carrondo, MJT
中科院分区：
文献类型：
--
作者：
Cunha, AE;Clemente, JJ;Carrondo, MJT
纤维连接蛋白剪接变异体ED B(细胞外域B)是一种有希望的实体肿瘤血管生成的标志物。目前，针对ED B的重组抗体正在研究其潜在用途，无论是用于治疗还是诊断目的。针对ED B的单链抗体片段可以在巴斯德毕赤酵母中高效表达，因此，本工作使用了一株重组的嗜甲基酵母巴斯德酵母。在发酵上清液中发现了三种不同形式的ScFv抗体片段：共价同源二聚体、缔合同源二聚体和单体。这两种同源二聚体形式都可以(在还原条件下)转化为单体形式并被有效地放射性标记，而上清液中已经存在的单体形式的单链抗体不能。研究还发现，单体形式的蛋白质比例高度依赖于诱导方式，而不是ScFv的浓度。这表明上清液中单链抗体的单体形式可能是在表达、分泌或折叠水平上发生的事件的结果。通过优化甲醇诱导，建立了高密度发酵工艺，获得了最高的ScFv抗体片段产量和产品质量，诱导点的细胞浓度和比甲醇摄取率是最重要的控制变量。比甲醇摄取率的降低导致单链抗体片段的比产量较高(5.4mug(Cell)h(-1))。产品质量，即以均二聚体形式存在的产品的百分比，也随着甲醇吸收率的降低而增加。此外，体积生产力依赖于诱导点的细胞浓度，随着细胞浓度的增加而增加，直到320g L湿细胞重(WCW)。降低用于诱导的甲醇进料速度，从而降低摄氧率，对于优化产品效价和质量，从而扩大生产过程具有重要的后果；因此，生物反应器中高细胞密度培养的主要限制之一是保持所需的高氧气转移速率。根据获得的结果，根据更大范围内的可用氧转移速率制定了放大策略，从而定义了诱导的最佳生物量浓度和最大化产品效价和质量的甲醇进料策略。(C)2004年威利期刊公司。
Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 mug g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L-1 wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality. (C) 2004 Wiley Periodicals, Inc.