Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.
Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.
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DOI：
10.1002/bit.28230
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发表时间：
2022-12
影响因子：
3.8
通讯作者：
Smales, C. Mark
中科院分区：
文献类型：
--
作者：
Knight, Tanya J.;Povey, Jane F.;Vito, Davide;Mohindra, Atul;Jaques, Colin M.;Smales, C. Mark
关键词：
哺乳动物细胞，特别是中国仓鼠卵巢细胞，是以蛋白质为基础的生物治疗药物的主要生产系统，然而，有时会观察到产物的降解，特别是Fc融合蛋白，影响所产生的蛋白质的质量。在这里，我们确定了一个模型免疫球蛋白G1Fc融合蛋白的碎片位置，表明低温培养减少了观察到的剪裁和聚集，碎片/剪裁是细胞内的，并且在较低的温度(<；37℃)下减少的剪裁与Mesenger RNA(MRNA)翻译延伸有关。我们随后证明，通过添加减缓平移延伸的化学试剂，可以在37℃下减少碎裂。然后，我们根据不同的密码子使用情况，通过为Fc-融合蛋白设计不同的转录序列来修改mRNA的翻译延伸速度，并在37℃下提高了产物产量和完整与片段产物的比率。我们的数据表明，快速的伸长会导致错误折叠，从而降低产品的保真度，产生一个容易降解/蛋白质降解的区域，而mRNA翻译的减慢则改善了折叠，降低了对碎片的敏感性。因此，操纵mRNA翻译和/或目标Fc-融合转录本是一种可以潜在地减少易于剪裁的Fc-融合蛋白的碎片的方法。
Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein‐based biotherapeutics, however, product degradation, particularly of Fc‐fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc‐fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc‐fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc‐fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping‐prone Fc‐fusion proteins.
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影响因子：
3.7
作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
10.1016/j.jchromb.2004.12.033
发表时间：
2005-04-25
影响因子：
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影响因子：
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通讯作者：
Mitchell-Logean, Christine
影响因子：
4.1
作者：
Roobol, Anne;Roobol, Jo;Smales, C. Mark
通讯作者：
Smales, C. Mark