Engineering an alkaline-stable protein A through rational design strategies.

In recent years, monoclonal antibody drugs have dominated the bio-pharmaceutical market because of their high specificity and low adverse reactions. Antibody purification has always occupied most of the cost of antibody drug production. Protein A affinity chromatogra phy, as the most commonly used antibody purification method, has been explored to improve its service life and reduce the cost of purification. Cleaning in place (CIP) of protein A requires protein A to have a better alkalinity resistance for protein A affinity chromatography reuse. Therefore, in this study, we redesigned a base-tolerant protein A, 4xPA-LPA, by semi-rational analysis based on bio-informatics. Including surface charge analysis, construction of linker peptides, and MSA and FoldX prediction-based conserved site screening and targeting mutagenesis. Analyzed by SDS-PAGE, differential scanning calorimeter and circular dichroism, the results showed that 4xPA-LPA was treated with 0.5 M NaOH and more than 84 % residues remain after 24 h. The Tm value of 4xPA-LPA increased by 2.47 °C compared with that before mutation. Its α-helices number increased, and the protein structure was more stable. DBC retention was approximately 86 % after alkali immersing and 89 % after 100 simulated wash repetitions. The novel alkali-resistant protein A has good application prospects in the field of monoclonal antibody purification, and this study also provides a certain reference strategy for future engineering to improve the alkali-resistance of proteins.