Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant...

Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.