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

Abstract

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.

Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells

“…We determined the expression at protein level of the recombinant immunoglobulin chains and found that the LC polypeptide was more abundant than the HC polypeptide (HC:LC ratio \1). This finding is consistent with previous reports suggesting that HC levels could limit productivity while LC excess is required for efficient antibody folding and assembly (Smales et al 2004;Schlatter et al 2005;Dorai et al 2006). During folding and assembly of IgG in the endoplasmic reticulum of mammalian cells, immunoglobulin chain polypeptides sequentially interact with a range of molecular chaperones, foldases, and oxidoreductases present in macromolecular complexes in the endoplasmic reticulum such as BiP (immunoglobulin binding protein) and PDI (protein disulfide isomerase) .…”

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

“…In the absence of the WPRE, the light to heavy chain mRNA ratio was nearly 1:1 at 31°C, while this ratio in the presence of the WPRE was about 5:1. It has previously been shown that the level of the light chain polypeptide is limiting for antibody assembly (35,36). Thus, the effect of the WPRE may be to increase the steady-state level of the light chain mRNA, resulting in higher light chain protein levels and higher antibody secretion.…”

Proteomic Analysis of Bioreactor Cultures of an Antibody Expressing CHOGS Cell Line that Promotes High Productivity

“…In an attempt to increase overall productivity of these cell lines, a number of investigations have been undertaken in the past several years. These investigations, which follow closely those published in the literature, include 1) determining the optimal copy numbers of H-ch and L-ch of the therapeutic Ab in the transfectomas [17,40]; 2) utility of apoptotic resistant host cell lines [13,23,24]; 3) increased secretion of Abs via over-expression of chaperones [3] (unpublished observation); 4) prolongation of the G1 phase where protein synthesis is particularly more efficient [25] and 5) judicious use of expression systems and cell line selection strategies [2,41,42]. Recently, the "omics" approach for The effect of four different media components on multiple cell culture parameters.…”