Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells

ABSTRACT

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.

KEYWORDS:

• Alternative splicing
• CHO
• expression plasmid
• intron
• monoclonal antibody
• splice variant

Abbreviations

CHO	=	Chinese hamster ovary
CSR	=	class switch recombination
CQA	=	critical quality attributes
GS	=	glutamine synthetase
HC	=	heavy chain
HCCD	=	heavy chain constant domain
IgG	=	immunoglobulin
IVC	=	integral of viable cells (109 cell hr/L)
LC	=	light chain
mAbs	=	monoclonal antibodies
qP	=	cell productivity (pg/(cell day))
VCN	=	viable cell number (x106/mL)
3′SS	=	3′ splice site

Acknowledgments

The authors would like to thank Fabio Zurlo and the Bioprocess assay team at AstraZeneca for performing HPLC titer analysis; Jie Zhu and Ying Liu for input into the design of the intron retention assay; Darren Geoghegan and Thomas Albanetti for initial work evaluating different heavy-chain gene constructs; and Luigi Grassi for support and helpful discussion.

Disclosure statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was supported by Biopharaceutical Development, AstraZeneca. Authors E.K., C.H., D.H., S.D., and S.G. are employees of AstraZeneca and have stock and/or stock interests or options in AstraZeneca.

Author contributions

All authors provided input into the research and manuscript. E.K., C.H., D.H., S.D., and S.G. conceptualized and designed the experiments; E.K. and T.S. performed experiments and analyzed the data and E.K., T.S., D.H., S.D., and S.G. wrote and reviewed the manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2242548

Additional information

Funding

The author(s) reported that there is no funding associated with the work featured in this article.