Exploiting ligand-driven salt selectivity for aggregate retention: enhancing monomer resolution on a high elution pH Protein A membrane

Protein A chromatography often requires product-compromising low-pH conditions to remove monoclonal antibody aggregates. While recent high-pH ligands and membrane architectures have shown to improve aggregate clearance, the underlying mechanism of how the ligand and the matrix each contribute to this separation remains unclear. Here, we evaluated a recombinant high-pH Protein A prototype ligand immobilized on a Natrix membrane and a bead-based resin. We found that the addition of NaCl across a wide concentration range substantially enhanced monomer-aggregate resolution in a dose-dependent manner. Elevated ionic strength caused a slight shift in monomer elution toward milder conditions while simultaneously driving the tight retention of the aggregate subpopulation. At concentrations ≥ 0.5 M NaCl, aggregates were retained so strongly that they required complete salt removal under low-pH conditions to elute. Comparative evaluation proved this divergent selectivity is intrinsically driven by the recombinant ligand's specific binding response to salt, rather than being a mass-transport artifact of the membrane format. Leveraging this mechanism, we developed a stepwise elution strategy achieving > 70 % aggregate reduction and > 90 % monomer yield at pH 5.0, independent of initial aggregate burden. Additionally, a HIC-like flow-through mode confirmed the preferential retention of higher-order multimers. Ultimately, this work demonstrates that modulating ionic strength on engineered ligands establishes a highly tunable, mild-pH strategy for the robust purification of aggregation-prone therapeutics.