title: Capto MMC mixed-mode chromatography of murine and rabbit antibodies.
abstract: Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0.
authors.affiliation: Alliance Protein Laboratories, 6042 Cornerstone Court West, San Diego, CA 92121, USA. Electronic address: tarakawa2@aol.com.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
authors.affiliation: Abwiz Bio, Inc., 9823 Pacific Heights BLVD, Suite J, San Diego, CA 92121, USA.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kurosawa
authors.affiliation: Abwiz Bio, Inc., 9823 Pacific Heights BLVD, Suite J, San Diego, CA 92121, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Storms
authors.affiliation: Abwiz Bio, Inc., 9823 Pacific Heights BLVD, Suite J, San Diego, CA 92121, USA.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Abwiz Bio, Inc., 9823 Pacific Heights BLVD, Suite J, San Diego, CA 92121, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Okumura
authors.affiliation: Alliance Protein Laboratories, 6042 Cornerstone Court West, San Diego, CA 92121, USA.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Kita
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D013205:Staphylococcal Protein A
keywords: Arginine; Capto MMC; Hydroxyapatite; Mixed-mode chromatography; Murine antibody