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On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells

机译:CHO细胞产生高效重组抗体的重链和轻链基因的最佳比例

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摘要

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides.Accordingly,most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (hc) to light chain (Zc) genes encoded on either one or two plasmids.However,there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab.In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG4 Mab,cB72.3,by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches.Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2;a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data.On the basis of this information,we hypothesized that utilization of hc genes at low hc:lc gene ratios is more efficient.To confirm this,cB72.3 Mab was transiently produced by CHO cells at constant hc and varying Zc gene dose.Under these conditions,Mab yield was increased with a concomitant increase in Zc gene dose.To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab,we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab,cB72.3.Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5,less than that observed on transient expression of the same Mab in parental CHO cells using the same vector.In conclusion,our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ.In the case of the latter,we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.
机译:单克隆抗体(Mab)是异四聚体,由等摩尔比的重链(HC)和轻链(LC)多肽组成。因此,大多数重组Mab表达系统利用等摩尔比的重链(hc)与轻链(Zc)基因编码在一个或两个质粒上。但是,没有证据表明该基因比例对于稳定或瞬时生产重组Mab而言是最佳的。在这项研究中,我们确定了hc:lc基因在重组质粒生产中的最佳比例。中国仓鼠卵巢(CHO)细胞使用经验和数学建模方法对IgG4 Mab,cB72.3进行研究。聚乙烯亚胺介导的cB72.3瞬时表达,在不同质粒上编码的hc:lc基因的不同比例产生的最佳Mab效价为hc:lc基因比例为3:2;通过使用瞬时表达数据对Mab折叠和组装过程进行单独的数学建模证实了这一结论。基于此信息,我们假设utiliz在低hc:lc基因比例下对hc基因进行更高效的鉴定。为证实这一点,CHO细胞在恒定hc和Zc基因剂量不同的情况下瞬时产生了cB72.3 Mab。在这些条件下,Mab产量随之增加。为了确定以上发现是否也适用于稳定转染的生产重组Mab的CHO细胞,我们比较了以1:1的转染率转染的三种GS-CHO细胞系的HC和LC多肽的细胞内和细胞外比率。 hc:lc基因,并选择用于稳定表达同一重组Mab,cB72.3。细胞内和细胞外HC:LC多肽比例为1:2至1:5,低于在同一Mab中瞬时表达时观察到的比例。总之,我们的数据表明,用于瞬时和稳定表达Mab的hc:lc基因的最佳比例有所不同。对于后者,我们推断稳定转染的细胞可产生最佳Mab表示妥协补间HC的丰度限制了生产率以及对过量LC的要求,以使Mab折叠和组装更有效。

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