Alternative splicing for tuneable expression of protein subunits at desired ratios

ABSTRACT

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Keywords:

• alternative splicing
• bicistronic expression
• tuneable subunit ratio
• ratio optimization
• controlled expression

Acknowledgments

The authors would like to thank Dr David Hacker for critically reading the manuscript and Dr Maria Tsachaki for valuable technical support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplemental material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2024.2342243

Additional information

Funding

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