Protein A affinity adsorbent with high antibody-binding capacity plays a prominent part in the purification of biopharmaceuticals to decrease the manufacturing costs. We describe a site-specific covalent conjugation strategy for protein A to immobilize on agarose beads. Recombinant protein A, which has one cysteine introduced at the C terminus through genetic engineering technology, was immobilized site-specifically on maleimide-functionalized agarose beads by the thiol–maleimide reaction. As a comparison, the recombinant protein A was randomly immobilized on the aldehyde-functionalized agarose beads via free amino groups on the protein surface. The site-specific conjugation of recombinant protein A on the agarose beads was validated through the assay of free SH groups on the adsorbents using the Ellman’s reagent. Adsorbents containing various amounts of protein A were used to adsorb antibody from human plasma. Analysis of immunoturbidimetry showed that the adsorbed fractions contained the 90.1% IgG, 4.2% IgA, and 5.7% IgM. The maximal antibodies-binding capacities with staticadsorption and dynamic adsorption were approximately 64 and 50 mg,respectively, per swollen gram for site-specifically conjugated adsorbentand 31 and 26 mg for randomly conjugated adsorbent. Remarkably, thehigh antibody-binding capacity for site-specifically conjugated adsorbentoutperformed the existing commercial protein A Sepharose (approximately30 mg/g). The orientation of a protein is crucial for its activityafter immobilization, and these results demonstrate that the site-specificallyconjugated protein molecule is in a functionally active form to interactwith the antibody with weak steric hindrance. The proposed approachmay be an attractive strategy to synthesize affinity adsorbents withhigh-binding capacity.
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