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

Abstract

Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins 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 to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can 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 ∼20-fold less DNA and ∼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 affinity chromatography method.

Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

“…After two rounds of the separation processes, the purity and recovery of the hanA1-EmGFP were estimated about 75% and 67%, respectively (Table 1), less than about 95% recovery of the ZZ-CSQ [7]. Recovery of the His6-eGFP in E. coli with Ni-NTA purification is up to about 63%.…”

“…Other fusion tags such as the ELP, hydrophobin and CspB are used for non-chromatographic purification under room temperature [6,32,33]. Different from the intein in the annexin fusion to potentially limit protein solubility [6], and the CSQ unable to be removed from the ZZ-CSQ [7], our constructs will be used for yielding the non-tagged target proteins via specific protease for cleaving the sequence introduced between the hanA1 and target protein, similar to the other proteases recognizing sequences incorporated between the ELP tag and target protein [34].…”

“…His6-tag and Strep-tag II, fused either to the hanA1 at N-terminus for forming the tandem affinity tag, or with the target protein at C-terminus to generate double affinity tag will further offer various purification strategies to obtain pure protein. The fluorescent and colored proteins selected in this study are ideal for testing the other tag applicability for Ca 2+ dependent separation or purification, such as CSQ [7], and Fh8 tag.…”

“…Based on the fluorescence measurement, nearly 80% hanA1-EmGFP was precipitated. In contrast, deletion of C-terminal 16 amino acid residues in the ZZ-CSQ allowed about 93% fusion protein to form a precipitate at 10 mM CaCl 2 within 10 min [7].…”

“…So, this tag is only utilized for purifying a few of peptides. Recently, the Ca 2+ dependent phase-transition properties of calsequestrin (CSQ) permit the protein fused with the Z domain of protein A (ZZ-CSQ) for affinity separation [7]. Developing the novel fusion tag at the N-terminal partner with high selectivity for simple, costeffective separation and purification is required for isolating recombinant protein and enzyme with industrial and clinical values.…”