An engineered Staphylococcal Protein A based ligand: Production, characterization and potential application for the...

In antibody purification processes, affinity chromatography has been used with Staphylococcus aureus protein A (SpA) as the main ligand. In this work, we present a novel Staphylococcal Protein A (AviPure thereafter), a synthetic ligand analogue based on native SpA B domain, with a molecular weight of approximately 14 kDa. The binding affinity of mAbs to AviPure was evaluated using Surface Plasmon Resonance (SPR) and affinity chromatography methods. The equilibrium dissociation constant (K_D) between the AviPure and mAbs was systematically measured using 1:1 (Langmuir) model and found to be 4.7 × 10^-8 M, with constant of dissociation at k_d ≤ 1.0 × 10^-3 s^-1 and k_a being 3.1 × 10⁴ M^-1 s^-1. When immobilized on Sepharose, the AviPure ligand density was 429 nmol/g moist weight resin and was able to effectively bind immunoglobulin and Fc fragment samples with higher affinity and the most effective flow rate when using ligand - Sepharose beads was at 75 cm/h giving the dynamic binding capacity of 53 mg/mL and 91% recovery of IgG. Suitable ligands used in affinity purification should have a K_D ≤ 10^-6 M and a dissociation rate (k_a) averaging 10^-3 M^-1 s^-1 with the k_d ranging between 10³ - 10⁸ M^-1. Therefore, the AviPure ligand can be used as an alternative to the standard protein A ligand in the purification of mAbs and Fc-fused proteins.