Purification of Therapeutic Antibodies Using the Ca2+-DependentPhase-Transition Properties of Calsequestrin

:affinity chromatography utilizing specific interac-tions between therapeutic proteins and bead-immobilized captur-ing agents is a standard method for protein purification, but itsscalability is limited by long purification times, activity loss by thecapturing molecules and/or purified protein, and high costs. Here,we report a platform for purifying therapeutic antibodies viaaffinity precipitation using the endogenous calcium ion-bindingprotein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusionproteins with CSQ and an affinity protein (Z domain of protein A)capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody tobe collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platformcan rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95%+/- 3%).In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding similar to 20-fold less DNA and similar to 4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable,scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinitychromatography method.

Automated summary

In this study, a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), is reported. The ZZ-CSQ fusion proteins capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. The platform is rapid, recyclable, scalable and cost-effective, and has better impurity removal performance compared to traditional protein A-based affinity chromatography.