Schlatter Stefan, Stansfield Scott H, Dinnis Diane M, Racher Andrew J, Birch John R, James David C
School of Engineering, University of Queensland, QLD 4072, Australia, and Lonza Biologics plc, 228 Bath Road, Slough SL1 4DX, UK.
Biotechnol Prog. 2005 Jan-Feb;21(1):122-33. doi: 10.1021/bp049780w.
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 (lc) 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 lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc 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.
单克隆抗体（Mab）是由等摩尔比的重链（HC）和轻链（LC）多肽组成的异源四聚体。因此，大多数重组单克隆抗体表达系统在一个或两个质粒上利用等摩尔比的重链（hc）和轻链（lc）基因。然而，没有证据表明这种基因比例对于重组单克隆抗体的稳定或瞬时生产是最佳的。在本研究中，我们使用经验和数学建模方法确定了中国仓鼠卵巢（CHO）细胞生产重组IgG4单克隆抗体cB72.3时hc:lc基因的最佳比例。通过聚乙烯亚胺介导在不同质粒上编码的hc:lc基因比例下瞬时表达cB72.3，当hc:lc基因比例为3:2时产生了最佳的单克隆抗体滴度；使用瞬时表达数据对单克隆抗体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，我们假设在低hc:lc基因比例下使用hc基因更有效。为了证实这一点，CHO细胞在恒定的hc和不同的lc基因剂量下瞬时产生cB72.3单克隆抗体。在这些条件下，随着lc基因剂量的增加，单克隆抗体产量增加。为了确定上述发现是否也适用于稳定转染的产生重组单克隆抗体的CHO细胞，我们比较了三个用1:1比例的hc:lc基因转染并选择稳定表达相同重组单克隆抗体cB72.3的GS-CHO细胞系的细胞内和细胞外HC和LC多肽的比例。细胞内和细胞外HC:LC多肽比例范围为1:2至1:5，低于使用相同载体在亲本CHO细胞中瞬时表达相同单克隆抗体时观察到的比例。总之，我们的数据表明用于单克隆抗体瞬时和稳定表达的hc:lc基因的最佳比例不同。对于后者，我们推断稳定转染细胞产生最佳单克隆抗体代表了限制生产力的HC丰度与过量LC使单克隆抗体折叠和组装更有效的要求之间的折衷。
