Rational design and engineering of protein A to obtain the controlled elution profile in monoclonal antibody purification

Biopharmaceutical monoclonal antibodies (Mabs) show different chromatographic behaviors in the elution step on protein A chromatography, although Mabs have similar three-dimensional structures. It is well known that interactions of conventional protein A to the VH3 subfamily variable region negatively affect Mabs elution properties. The mutation G29A is known to weaken this binding, although not always sufficiently. We designed novel protein A mutations, S33E and D36R, by a computer-aided evaluation based on the three-dimensional structure. These mutations are expected to not only eliminate protein A binding to the variable region of Mabs but also to maintain its alkaline stability, which is required for effective CIP (Clean in place) of the protein A affinity matrix. In view of the superior potential of C domain, an in vitro study was performed with the G29A mutant of C domain (C-G29A) as a model protein. Both pentameric C domain mutants (C-G29A/S33E.5d and C-G29A/D36R.5d) showed little binding ability to the VH3 subfamily variable region of Mabs by BIACORE analysis. We used a C-G29A/S33E.5d-immobilized matrix to confirm that the elution profile of Mabs belonging to the VH3 subfamily at pH 3.5 was significantly improved. This matrix also showed almost the same alkaline stability as did the C-G29A.5d-immobilized matrix. The engineered protein A ligand, whose binding ability to the variable region is completely eliminated, would enable the separation of Fab fragments in flow-through fractions from Mab digestions. Rational design by a computer-aided evaluation should enhance the efficiency of protein ligand engineering.