Optimizing antibody purification is crucial to overcoming a bottleneck in the costly manufacturing process for antibody therapy. To address this issue, we designed a pH-sensitive Staphylococcus aureus protein A variant that retained its innate stability and affinity toward antibody. On the basis of structural information and mutation analysis data, we identified candidate positions for accumulative histidine substitutions to cause electrostatic repulsion under acidic conditions. The histidine substitutions effectively decreased the dissociation rate under acidic conditions by three orders of magnitude. Avoiding deleterious effects of the substitutions, we successfully engineered a protein A variant that exhibited high pH sensitivity and maintained affinity, thermal stability, and alkaline tolerance. The variant was capable of serving as an affinity ligand that made affinity chromatography under milder acidic conditions possible; the elution peak shifted from pH 4.2 to 5.6. Only two substitutions were needed to achieve this pH sensitivity. This structure-based approach is applicable to other protein-based ligands.
Submitter: Shu-Ching Ou
Submission Date: Nov. 22, 2018, 8 a.m.
|Number of data points||20|
|Proteins||Immunoglobulin G-binding protein A|
|Assays/Quantities/Protocols||Experimental Assay: Elution peak: Affinity chromatography ; Experimental Assay: Binding: koff ; Experimental Assay: Alkaline Tolerance: Remaining Binding Activity ; Experimental Assay: Thermal Stability: Tm ; Experimental Assay: Binding: Kd|
|Libraries||Variants for Complex PAZST ; Variants for Protein A ; Variants for Complex CM5 ; Variants for Complex SA|