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

详细信息    查看全文

• 作者：Stefan Schlatter ; Scott H. Stansfield ; Diane M. Dinnis ; Andrew J. Racher ; John R. Birch ; and David C. James
• 刊名：Biotechnology Progress
• 出版年：2005
• 出版时间：February 2005
• 年：2005
• 卷：21
• 期：1
• 页码：122 - 133
• 全文大小：718K
• 年卷期：v.21,no.1(February 2005)
• ISSN：1520-6033

文摘

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio ofheavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinantMab 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 tosuggest that this gene ratio is optimal for stable or transient production of recombinantMab. In this study we have determined the optimal ratio of hc:lc genes for productionof a recombinant IgG₄ Mab, cB72.3, by Chinese hamster ovary (CHO) cells using bothempirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmidsyielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed byseparate mathematical modeling of the Mab folding and assembly process usingtransient expression data. On the basis of this information, we hypothesized thatutilization of hc genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3Mab 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 lcgene dose. To determine if the above findings also apply to stably transfected CHOcells producing recombinant Mab, we compared the intra- and extracellular ratios ofHC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio ofhc: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 thanthat observed on transient expression of the same Mab in parental CHO cells usingthe same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genesused for transient and stable expression of Mab differ. In the case of the latter, weinfer that optimal Mab production by stably transfected cells represents a compromisebetween HC abundance limiting productivity and the requirement for excess LC torender Mab folding and assembly more efficient.