Optimizing assembly and production of native bispecific antibodies by codon de-optimization

Abstract

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. kλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a kλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the kλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.

Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies

“…We have shown in our previous study that the effects on light chain content and IgG form distribution obtained with different de-optimized variants were consistent between transient transfections using PEAK cells and expression from stable CHO pools [13]. In this study, the results obtained with both systems were also similar for K2O41 but appeared to be different for K2O30.…”

“…We selected these three κλ bodies as they displayed different degrees of lambda chain overexpression and we aimed at restoring a more balanced expression via a codon de-optimization approach [13]. However, to avoid designing several new sequences for each candidate as previously described, we evaluated the possibility to achieve down modulation while restricting the sequence modification to the constant region of the lambda chain.…”

“…As for other formats that rely on the co-expression of multiple chains, κλ body yield as well as abundance of by-products is affected by the expression and assembly rates of the different polypeptides. Thus, it is important to aim at controlling their relative expression levels, which can be achieved by altering gene copy number, promoter strength or by DNA sequence optimization [13,18,19]. We previously described that the introduction of lower frequency codons into the sequence encoding both the variable and constant domains of a highly expressed light chain could significantly improve bsAb yield.…”

“…However, beyond discovery and bsAb characterization, when considering large scale expression and manufacturing, maximizing yield via fine tuning the expression of different chains might become particularly important. We previously demonstrated that altering the expression of one light chain could significantly shift the distribution between bsAb and by-products [13]. Interestingly, reducing the expression of the most abundant light chain was significantly more effective than optimizing the codon usage to increase expression of the underrepresented light chain.…”

Design and Production of Bispecific Antibodies

“…Columns specific for kappa- and lambda-monospecific antibodies isolation were then adopted followed by Protein A purification, although only around 50% of the final product is κλ body with the rest mainly including kappa-kappa and lambda-lambda antibody side products [140,164]. Interestingly, another research group reported that codon de-optimization of the lambda chain sequence increased the κλ body yield two-fold and enhanced the relative distribution of bispecific antibodies in a low kappa chain expressing κλ body cell line [165].…”