Scientists develop gentler way to purify antibody drugs using calcium
Researchers have engineered a new protein that lets manufacturers capture and release antibody therapeutics using calcium instead of harsh acids, potentially cutting production costs and time. The breakthrough could accelerate the race to bring antibody fragment drugs to market, a growing segment of the biopharmaceutical industry.
Originaltitel: Calcium as a molecular switch: gentle antibody-purification through directed evolution of Protein G
<p>Antibody fragments are emerging as promising therapeutics, with biomolecular recognition properties on par with full-length monoclonal antibodies, while also providing advantages in terms of biodistribution and manufacturability. While antigen-binding fragments are generally easy to produce, with a variety of expression systems available, the downstream processing has faced challenges due to the lack of a mild selective capture approach for purification. This study addresses the challenge by demonstrating that directed evolution of a Protein G-derived antibody-binding domain into a calcium-dependent ligand, C2<sub>Ca</sub> <sub>EP7</sub>, that enables mild capture and elution of full-length mouse IgG1 by the interaction with the Fab-fragment. The evolved ligand allows efficient purification of mouse IgG1 with calcium-triggered elution at neutral pH, eliminating the need for acidic conditions typically used in affinity chromatography. Optimization studies demonstrate that while the monomeric format elutes antibodies at moderate salt concentrations, a tetrameric configuration enhances binding strength (in 1 mM CaCl<sub>2</sub>) through avidity effects and enables near-complete antibody recovery without leakage using 150 mM NaCl and 100 mM citrate for elution. Mechanistically, solution-state NMR reveals that a unique E35G scaffold mutation, acting synergistically with loop modifications, destabilizes the domain in the absence of calcium, leading to loss of tertiary structure and disruption of the antibody-binding interface, while calcium binding restores the native conformation and functional binding surface. Collectively, these results establish a calcium-switchable affinity system that enables robust, mild, and scalable antibody purification at neutral pH without residual product remaining on the column.</p>