Abstract：
In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein\'s primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.
摘要：
体外蛋白质重折叠是制备融合蛋白(例如使用大肠杆菌表达的肽体)中的关键限速单元操作之一。稀释辅助重折叠是最常用的工业实践，以实现可溶性，来自包涵体的重组蛋白的天然功能形式。这项研究的重点是开发一种色谱辅助的体外重折叠平台，以产生生物活性，重组肽体的天然形式。选择重组Romiplostim作为研究的模型蛋白。将塞流管式反应器与捕获步骤亲和色谱法串联连接，以同时实现重组Romiplostim的体外重折叠和捕获步骤纯化。各种关键工艺参数的影响，如倍数稀释，温度,停留时间,使用基于中心复合物的实验策略设计来研究半胱氨酸:DTT比率,以实现所选择的肽体的最大重折叠产率。在最佳重折叠条件下，在25倍稀释时达到57.0±1.5%的最大重折叠产率和超过79.73±3.4%的纯度，温度15°C,6小时的停留时间与6mM和10mM的半胱氨酸和DTT，分别。使用各种正交分析工具检查天然肽体结构的形成，以研究蛋白质的初级，次要,和三级结构。使用碰撞诱导解离(CID)对二硫键连接的肽的氨基酸序列进行定位,以确认Cys7-Cys7和Cys10-Cys10之间的链内二硫键的形成,类似于Cys42-Cys102和Cys148-Cys206之间的链内二硫键。这里开发的方案是鉴定涉及域间二硫键的多域蛋白的高产率可扩展重折叠条件的有价值的工具。
