Haryadi Ryan, Ho Steven, Kok Yee Jiun, Pu Helen X, Zheng Lu, Pereira Natasha A, Li Bin, Bi Xuezhi, Goh Lin-Tang, Yang Yuansheng, Song Zhiwei
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
PLoS One. 2015 Feb 23;10(2):e0116878. doi: 10.1371/journal.pone.0116878. eCollection 2015.
Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.
由信号肽介导的新生蛋白质从细胞质转运到内质网是蛋白质分泌中的关键步骤。这项工作的目的是开发一种平台技术，以优化信号肽，从而在CHO细胞中高水平生产治疗性抗体。构建了一个包含大量人类免疫球蛋白（Ig）重链（HC）和κ轻链（LC）信号肽的数据库。大多数HC信号肽包含19个氨基酸，可分为三个结构域，而LC信号肽包含22个氨基酸。然后根据序列相似性对信号肽进行聚类。基于聚类结果，分析了8个HC信号肽和2个LC信号肽对5种最畅销抗体药物（即赫赛汀、阿瓦斯汀、类克、利妥昔单抗和修美乐）生产的影响。确定了生产这5种抗体的最佳HC和LC信号肽。与公共数据库中可用的利妥昔单抗天然信号肽相比，优化后的信号肽生产效率提高了2倍。阿瓦斯汀优化后的HC信号肽中单个氨基酸的替换显著降低了其产量。质谱分析表明，所有优化后的信号肽在成熟抗体中均被准确切除。本报告中的结果对于这5种抗体作为生物类似药的生产尤为重要。它们也有可能成为CHO细胞中生产新抗体的最佳信号肽。
