(157f) Strategic Non-Optimal Codon Usage Reduces Bispecific Protein Mispairing during Transient Protein Expression

Multispecific biologics are a promising new modality for protein therapeutics, yet problems with proper expression and purification can hinder their development. Specifically, imbalanced component chain expression can lead to the assembly of mispaired species that need to be purified away from the final product.

We report here on the use of codon optimization as a method to improve bispecific protein expression. While codon optimization generally seeks to avoid non-optimal codon usage to accelerate protein translation, we found that deliberately incorporating non-optimal codons into the coding sequence of certain heavy and light chains can increase final product purity after a single-step Protein A purification from around 60% to >95%. We also find that this increase in purity is negatively correlated with total purified protein quantity. Current strategies for tuning chain expression ratios primarily focus on mRNA transcription, either through tuning promoter strength or plasmid transfection ratios. Our work suggests that it may be possible to optimize chain expression through codon usage without altering transcript levels.

By deliberately incorporating non-optimal codons into codon-optimized therapeutic protein coding sequences, our work demonstrates that strategic codon ‘de-optimization’ can lead to a higher quality final product.