Tuning the Assembly of Bispecific Antibodies by Playing on Differential Polypeptide Chain Molar Ratios

• Published in: Biotechnology and bioprocess engineering Vol. 28; no. 4; pp. 658 - 671
• Main Authors: De Temmerman, Célia, Bernard, Pauline, Aeberli, Quentin, Braito, Sarah, Arib, Ghislaine, Girod, Pierre-Alain, Fagète, Séverine
• Format: Journal Article
• Language: English
• Published: Seoul The Korean Society for Biotechnology and Bioengineering 01.08.2023; Springer Nature B.V; 한국생물공학회
• Subjects: Antibodies; Assembly; Biotechnology; Bispecific antibodies; cell lines; Chemistry; Chemistry and Materials Science; class; Cloning; Cricetulus griseus; dimerization; genes; Industrial and Production Engineering; mechanism of action; Mode of action; Polypeptides; Production methods; Protein engineering; Protein purification; Research Paper; Steering; therapeutics; 생물공학; bispecific antibody; titer; assembly; oriented expression; molar ratios; heterodimer
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-023-0057-9

Bispecific antibodies (bsAb) represent the most promising class of therapeutics due to their multiple mode of action. Their development raised tremendous efforts with a special focus on the engineering of these complex molecules, especially when they adopt an asymmetrical format. Various strategies have been successful in improving heterodimerization through the introduction of point mutations to force the correct pairing of opposite chains using either steric, electrostatic steering effects or both. These great advances in protein engineering combined with the development of robust process purification methods to remove homodimers have contributed to the fast progress of bsAbs towards clinics. Nevertheless, there is still a lack of systematic and efficient production methods to fine tune the expression of correctly paired bsAbs. In this study, we have evaluated the effects of manipulating different molar ratios of three polypeptide chains constituting a model bsAb, co-transfected, expressed and assembled in a single Chinese Hamster Ovary cell line. Two parental clones showing different heterodimerization and productivity profiles were chosen. We demonstrated that addition of gene copies at specific ratios to these clones could bias towards the generation of sub-clones with either reduced proper assembly or tune expression for enhanced integral bsAb. Our results demonstrate that expression of bsAb can be influenced beyond the initial engineering stage.