title: Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.
abstract: Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.
authors.affiliation: Department of Protein Analytical Chemistry, ‡Department of Biological Technologies, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hosken
authors.affiliation: Department of Protein Analytical Chemistry, ‡Department of Biological Technologies, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Charlene
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Protein Analytical Chemistry, ‡Department of Biological Technologies, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Berny
authors.initials: B
authors.lastname: Mullappally
authors.affiliation: Department of Protein Analytical Chemistry, ‡Department of Biological Technologies, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Carl
authors.initials: C
authors.lastname: Co
authors.affiliation: Department of Protein Analytical Chemistry, ‡Department of Biological Technologies, Genentech , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Boyan
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification* / Q000627:therapeutic use; D005591:Chemical Fractionation; D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing
chemical_list: D000911:Antibodies, Monoclonal