Christel Aebischer-Gumy, Pierre Moretti, Timothee Brunstein Laplace, Jana Frank, Ysaline Grand, Farid Mosbaoui, Emilie Hily, Anna Galea, Megane Peltret, Carole Estoppey, Daniel Ayoub, Roberto Giovannini, Martin Bertschinger
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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.","PeriodicalId":5,"journal":{"name":"ACS Applied Materials & Interfaces","volume":"7 26","pages":""},"PeriodicalIF":8.3000,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Alternative splicing for tuneable expression of protein subunits at desired ratios\",\"authors\":\"Christel Aebischer-Gumy, Pierre Moretti, Timothee Brunstein Laplace, Jana Frank, Ysaline Grand, Farid Mosbaoui, Emilie Hily, Anna Galea, Megane Peltret, Carole Estoppey, Daniel Ayoub, Roberto Giovannini, Martin Bertschinger\",\"doi\":\"10.1080/19420862.2024.2342243\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"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. 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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.
期刊介绍:
ACS Applied Materials & Interfaces is a leading interdisciplinary journal that brings together chemists, engineers, physicists, and biologists to explore the development and utilization of newly-discovered materials and interfacial processes for specific applications. Our journal has experienced remarkable growth since its establishment in 2009, both in terms of the number of articles published and the impact of the research showcased. We are proud to foster a truly global community, with the majority of published articles originating from outside the United States, reflecting the rapid growth of applied research worldwide.