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

Area of Science:

• Biotechnology
• Protein Engineering
• Biopharmaceutical Manufacturing

Background:

• Multi-specific antibody formats, such as Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT) antibodies, present expression challenges compared to traditional monoclonal antibodies (mAbs).
• The inherent complexity of these engineered proteins can lead to low expression titers, hindering their therapeutic development and manufacturing.

Purpose of the Study:

• To address and overcome the expression limitations associated with difficult-to-express bi-specific bi-paratopic BEAT antibodies.
• To develop a robust and scalable platform process for enhancing the production of complex multi-specific antibodies.

Main Methods:

• Investigated subunit misbalance as a root cause for poor expression.
• Optimized signal peptides and employed dual vector design elements on a single gene vector.
• Enhanced cell population selection, implemented an upstream (USP) platform process, and utilized a cell culture temperature shift.
• Developed a high-density seeding process for intensified cell culture.

Main Results:

• Achieved titer levels of up to 6 g/L for difficult-to-express BEAT antibodies through initial optimization.
• Increased titers to approximately 11 g/L for the BEAT antibody by implementing a high-density seeding process, representing a tenfold improvement.
• Successfully applied the developed platform to a tri-specific antibody, reaching titer levels of 10 g/L.

Conclusions:

• A comprehensive platform process integrating molecular biology, cell line development, upstream process optimization, and process intensification effectively enhances the expression of challenging multi-specific antibodies.
• The developed strategies provide a scalable solution for producing complex antibody formats, paving the way for their broader therapeutic application.