Abstract

Recently, there has been a high demand for anti-tumor necrosis factor-α monoclonal antibodies (mAbTNFα) in the treatment of rheumatoid arthritis and other autoimmune diseases. Thus, efficient strategies and stable high-producing cell lines need to be established to increase antibody production. In this study, we describe an efficient approach to establish a mAbTNFα high-producing clone through the optimization of expression vectors and cell culture media. The ubiquitous chromatin opening element (UCOE) and dihydrofolate reductase (DHFR)-based vectors encoding mAbTNFα were introduced into the CHO-DG44 cells using lipofection. Clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate, and isolating them by limiting dilution. Different media formulated with commercial feeds and media were also screened to develop an improved medium. The antibody produced by the selected clone was purified, characterized, and compared to standard adalimumab. Using our established protocol, a cell clone obtained from stable mAbTNFα-expressing cell pools showed a 3.8-fold higher antibody titer compared to stable cell pools. Furthermore, the highest antibody yield of selected clones cultured in fed-batch mode using improved medium was 2450 ± 30 µg/mL, which was 13.2-fold higher than that of stable cell pool cultivated in batch mode using a basal medium. The purified antibody had primary chemical and biological characteristics similar to those of adalimumab. Therefore, the use of UCOE and DHFR vectors in combination with the optimization of cell culture media may help in establishing stable and high-producing CHO cell lines for therapeutic antibody production.

摘要

近年来，抗肿瘤坏死因子-α单克隆抗体（mAbTNFα）在治疗类风湿性关节炎等自身免疫性疾病方面的需求量很大。因此，需要建立有效的策略和稳定的高产细胞系来增加抗体产量。在本研究中，我们描述了一种通过优化表达载体和细胞培养基来建立 mAbTNFα 高产克隆的有效方法。使用脂转染将普遍存在的染色质开放元件 (UCOE) 和基于二氢叶酸还原酶 (DHFR) 的编码 mAbTNFα 的载体引入 CHO-DG44 细胞。通过选择用 G418 转染的细胞、用甲氨蝶呤处理扩增它们并通过有限稀释分离它们来获得克隆。还筛选了用商业饲料和培养基配制的不同培养基以开发改进的培养基。对所选克隆产生的抗体进行纯化、表征，并与标准阿达木单抗进行比较。使用我们既定的方案，从稳定的 mAbTNFα 表达细胞池中获得的细胞克隆显示出比稳定细胞池高 3.8 倍的抗体滴度。此外，使用改良培养基以分批补料模式培养的选定克隆的最高抗体产量为 2450 ± 30 µg/mL，比使用基础培养基以分批模式培养的稳定细胞池高 13.2 倍。纯化的抗体具有与阿达木单抗相似的主要化学和生物学特征。所以，