A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.
A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.
复制标题
中国仓鼠卵巢细胞中重组 Fab 和全长抗体生产的比较分析。
DOI：
10.1002/bit.27944
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发表时间：
2021
影响因子：
3.8
通讯作者：
Hussain H
中科院分区：
文献类型：
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作者：
Hussain H
就批准用于治疗目的的数量而言，单克隆抗体是领先的生物制药类别。抗原结合片段 (Fab) 也用作生物治疗药物并广泛用于研究应用。全长抗体的主要表达系统是基于哺乳动物细胞的，而 Fab 分子的首选是在细菌系统中表达。然而，CHO 和下游技术的进步使哺乳动物系统成为小型和大规模 Fab 生产的同等可行的选择。使用一组全长 IgG 抗体及其具有不同抗原特异性的相应 Fab 对，我们研究了 IgG 和 Fab 分子格式对中国仓鼠卵巢 (CHO) 细胞生产的影响，并评估了细胞处理和生产这些格式的能力。与相应的 Fab 片段格式相比，全长抗体格式导致转染后恢复的迷你库更少，这可以解释为细胞总体负担更大。恢复后的抗体产生细胞池随后能够实现比相应 Fab 池更高的体积蛋白质产量 (mg/L) 和比生产率。重要的是，当考虑给定格式的每个细胞产生的实际分子（而不是质量）时，CHO 细胞每个细胞产生的 Fab 分子数量比用相应的 IgG 获得的要多，这表明细胞在制造较小的 Fab 分子方面更有效。细胞池分析表明，基因拷贝数与随后的蛋白质产生无关。 mRNA 的量与分泌的 Fab 产生相关，但与 IgG 无关，因此转录后过程会限制抗体的产生。总之，我们首次比较描述了全长 IgG 和 Fab 抗体形式如何影响细胞系构建过程的结果，并确定了其生产中的潜在限制，这些限制可用于提高这些重组抗体形式的生产效率。
Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen‐binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full‐length antibodies are mammalian cell‐based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small‐ and large‐scale Fab production. Using a panel of full‐length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full‐length antibody format resulted in the recovery of fewer mini‐pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody‐producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full‐length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.