title: Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions.
abstract: BACKGROUND
  In antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions.
  
  
  RESULTS
  Staphylococcal protein A variants were engineered by using both structure-based design and combinatorial screening methods. The frequency of amino acid residue substitutions was statistically analyzed using the sequences isolated from a histidine-scanning library screening. The positions where the frequency of occurrence of a histidine residue was more than 70% were thought to be effective histidine-mutation sites. Consequently, we identified PAB variants with a D36H mutation whose binding of IgG was highly sensitive to pH change.
  
  
  CONCLUSIONS
  The affinity column elution chromatograms demonstrated that antibodies could be eluted at a higher pH (∆pH**≧2.0) than ever reported (∆pH = 1.4) when the Staphylococcal protein A variants developed in this study were used as affinity ligands. The interactions between Staphylococcal protein A and IgG-Fab were shown to be important for the behavior of IgG bound on a SpA affinity column, and alterations in the affinity of the ligands for IgG-Fab clearly affected the conditions for eluting the bound IgG. Thus, a histidine-scanning library combined with a structure-based design was shown to be effective in engineering novel pH-sensitive proteins.
authors: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan ; Manufacturing Technology Association of Biologics, Chuo-ku, Kobe, Japan.
authors: Masayuki
authors: M
authors: Tsukamoto
authors: Biomedical Research Institute National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors: Hideki
authors: H
authors: Watanabe
authors: Biomedical Research Institute National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors: Ayako
authors: A
authors: Ooishi
authors: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan ; Biomedical Research Institute National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305-8566, Japan ; Manufacturing Technology Association of Biologics, Chuo-ku, Kobe, Japan.
authors: Shinya
authors: S
authors: Honda
keywords: Affinity purification; Antibody; Combinatorial screening; Histidine-scanning Library; Staphylococcal protein A