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 V H 3 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 V H 3 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 V H 3 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. -9.0 T (Thr) +23.9 (-3.3) +0.9 +15.4 (-9.9) -5.0 The results of the mutations at residue Ser-33 or Asp-36 of C domain are shown. As for the complex, another result by adjusting the relatively high dielectric constant is shown in parentheses ( ). The negative  G of the mutants (Fab complex or free-state) indicate better thermostability than the case of the respective wild type. Hence, in this study, the positive  G complex is preferable in a complex to eliminate the binding to Fab, while the negative  G free is preferable in the free