An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Cell-free protein synthesis (CFPS) enables antibody production, including those with non-natural amino acids for antibody drug conjugates (ADCs).
• Achieving high titers and robust assembly of complex proteins like IgGs in CFPS remains a challenge.

Purpose of the Study:

• To develop a scalable method for increasing IgG titers in cell-free systems.
• To enhance the robustness of cell-free antibody production.
• To enable efficient production of homogeneous antibody drug conjugates (ADCs).

Main Methods:

• A hybrid approach combining cell-based pre-expression of IgG light chains (LC) in an oxidizing E. coli cytoplasm with cell-free synthesis of heavy chains (HC).
• The prefabricated LC was added as a reagent to the XpressCF+® cell-free system for IgG assembly.
• Tested across various scales (100-µL to 0.25-L) and for different IgG formats, including bispecific antibodies.

Main Results:

• Achieved a 50-100% increase in IgG titers compared to traditional cell-free methods.
• Demonstrated increased robustness by eliminating the need to optimize HC-to-LC plasmid ratios.
• ADCs produced using this hybrid method showed comparable quality, in vitro cell killing, and FcRn binding to those made solely in cell-free systems.

Conclusions:

• Integrating in vivo and in vitro protein production significantly improves IgG titers and robustness in cell-free synthesis.
• This hybrid technology is a scalable and generalizable approach for producing complex antibodies and homogeneous ADCs.
• The method facilitates the production of site-specifically conjugated ADCs using non-natural amino acids.