Development of a 10 g/L process for a difficult-to-express multispecific antibody format using a holistic process development approach

• Published in: Journal of biotechnology Vol. 389; pp. 30 - 42
• Main Authors: Peltret, Mégane, Vetsch, Patrick, Farvaque, Elodie, Mette, Romain, Tsachaki, Maria, Duarte, Lionel, Duret, Anaïs, Vaxelaire, Emilie, Frank, Jana, Moritz, Benjamin, Aillerie, Céline, Giovannini, Roberto, Bertschinger, Martin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 20.06.2024
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Bispecific antibody; CHO Cells; Cricetulus; Difficult-to-express; Expression; Humans; Multispecific antibody; Difficult-to-express; Bispecific antibody; Expression; Multispecific antibody
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2024.04.017

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification. •Holistic development overcoming expression limits in bi-/multispecific antibodies via mol. bio., cell line dev, process opt.•Molecular constructs in article enable very high expression levels, particularly beneficial for challenging proteins.•Report of a process allowing titers > 10 g/L (product) for bi- and multi-specific antibody formats.