Investigation of alkaline effects on Protein A affinity ligands and resins using high resolution mass spectrometry

In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor (TM) plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe (TM) and MabSelect (TM) PrismA affinity chromatography (AC) resins after continuous incubation in 0.1-2.0 M NaOH for 1-72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand.

Automated summary

The study demonstrates the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches. MabSelect SuRe resin shows higher remaining binding capacity than MabSelect PrismA resin, while PrismA affinity ligand displays extreme alkaline stability even after incubation in 1.0 M NaOH for 72 hours.