mRNA stability and antibody production in CHO cells: Improvement through gene optimization

Abstract

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.

Generation of monoclonal antibody-producing mammalian cell lines

“…Codon optimization for gene expression is important due to differences in transfer RNA abundance in different cells and the effects on mRNA stability and second-ary structures formed by the transcripts [82][83][84]. Early attempts at codon optimization for gene expression improved green fluorescence protein (GFP) expression in CHO cells by 42-fold using the empirical method of DNA shuffling [85].…”

“…A comprehensive assessment of codon usage was performed in a recent proteomic analysis of CHO cells, and preference for certain codons was observed to be especially prevalent for proline, threonine, aspartate and cysteine [77]. Applying optimization based on codon usage in CHO and human cells has seen improvements in mAb expression of 1.5-4-fold [84,86]. A combination of codon optimization and an optimized gene amplification protocol provides a way to obtain high-yield mAb producers with decreased effort [87].…”

Why Proteins in Mammalian Cells?

“…Rather the removal of cryptic splice sites, minimizing secondary structures, balancing GC/AT richness, etc., are part of "gene optimization" (35,49).…”

The codon-optimized Δ6-desaturase gene of Pythium sp. as an empowering tool for engineering n3/n6 polyunsaturated fatty acid biosynthesis

“…To manipulate the oil composition of organisms of choice, by a metabolic engineering approach, a high expression level of the Δ 6 -desaturase enzyme in a heterologous host is required. As well as optimized culture conditions, an efficient promoter and substrate availability, codon optimization is one of the most common approaches for improving heterologous gene expression in some host organisms [8][9][10][11][12][13]. Although genes encoding for Δ 6 -desaturase have been cloned and characterized from various organisms [14][15][16][17][18][19][20], research on codon optimization has been limited [13].…”