Difficult-to-express antigen generation through a co-expression and disassociation methodology

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Extracellular domain (ECD) antigens are vital for therapeutic antibody development, including discovery, in vitro assays, and epitope mapping.
• Producing functional and stable ECD antigens using conventional expression systems often proves challenging, hindering therapeutic advancements.

Purpose of the Study:

• To develop and demonstrate an effective method for producing high-quality, biologically active extracellular domain (ECD) antigens, particularly those that are difficult to express.
• To overcome limitations of standard protein production techniques for complex antigens.

Main Methods:

• A novel co-expression strategy involving transiently co-expressing the antigen ECD with a stabilizing monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region in Chinese hamster ovary (CHO) cells.
• Post-expression, the antibody:antigen complex is purified, dissociated using low pH treatment with arginine, and the individual components are separated via preparative size exclusion chromatography (pSEC), with optional affinity chromatography for residual chaperone removal.

Main Results:

• Successfully produced stable and biologically active ECD antigens for two challenging targets from the cluster-of-differentiation/cytokine families, where traditional methods (Histidine-tagging, Fc-fusion) failed.
• Demonstrated the efficacy of the antibody-chaperone mediated co-expression and dissociation methodology for generating difficult-to-express antigens.

Conclusions:

• The antibody-chaperone mediated co-expression/dissociation method provides a robust and generalizable approach for producing stable, functional extracellular domain (ECD) antigens.
• This technique offers a viable solution for overcoming production challenges with antigens critical for therapeutic antibody development.