Identification of novel small molecule enhancers of protein production by cultured mammalian cells
Identification of novel small molecule enhancers of protein production by cultured mammalian cells
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DOI：
10.1002/bit.21839
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发表时间：
2008-08-15
影响因子：
3.8
通讯作者：
Reddy, Pranhitha
中科院分区：
文献类型：
--
作者：
Allen, Martin J.;Boyce, James P.;Reddy, Pranhitha
细胞培养基中的小分子添加剂（例如丁酸钠）能够增强重组蛋白的表达，在治疗性多肽的生产和制造中具有重要用途。为了在中国仓鼠卵巢（CHO）细胞中鉴定新型的重组蛋白表达小分子增强剂（SME），我们针对能够增强荧光报告蛋白或单克隆抗体表达的化合物，筛选了两个独立的小分子文库。鉴定出了几种能增加重组蛋白表达的化合物，这些化合物可分为三大类：（1）芳香族羧酸，（2）异羟肟酸，（3）乙酰胺。我们研究了向表达不同类别重组蛋白（包括单克隆抗体）的CHO细胞培养物中添加SME的影响。对于在生产摇瓶或生物反应器中生长的CHO细胞池或克隆，观察到重组蛋白滴度比对照培养物高出多达60%。对mRNA水平的分析表明，转录激活在某些SME所观察到的表达增强中起作用，但至少对于一种化合物可能涉及其他机制。最后，我们测试了许多已鉴定的SME提高杂交瘤细胞系单克隆抗体产量的能力。己基异羟肟酸相对于对照使摇瓶单克隆抗体产量提高了40%。综上所述，这些数据证明了这些化合物在从不同的基于细胞的生产系统生产治疗相关蛋白方面的潜在用途。
Small molecule additives to cell culture media (e.g., sodium butyrate) that are capable of enhancing the expression of recombinant proteins have significant utility in the production and manufacture of therapeutic polypeptides. To identify novel small molecule enhancers (SMEs) of recombinant protein expression in Chinese Hamster Ovary (CHO) cells, we screened two separate small molecule libraries for compounds capable of enhancing the expression of either a fluorescent reporter protein or a monoclonal antibody. Several compounds that increased recombinant protein expression were identified, and these compounds fell into three broad classes: (1) aromatic carboxylic acids, (2) hydroxamic acids, and (3) acetamides. We examined the impact of SME addition to CHO cell cultures expressing different classes of recombinant proteins including monoclonal antibodies (MAbs). For CHO cell pools or clones grown in production shake-flasks or bioreactors, recombinant protein titers up to 60% higher than control cultures were observed. Analysis of mRNA levels suggest that transcriptional activation plays a role in the expression enhancement seen for some SMEs, but other mechanisms may be involved for at least one compound. Finally, we tested many of the identified SMEs for their ability to increase MAb production by a hybridoma cell line. Hexanohydroxamic acid increased shake-flask MAb production by 40% relative to a control. Taken together, these data demonstrate the potential utility of the compounds in the production of therapeutically relevant proteins from diverse cell-based production systems.