title: Characterization of Chain Pairing Variants of Bispecific IgG Expressed in a Single Host Cell by High-Resolution Native and Denaturing Mass Spectrometry.
abstract: Bispecific antibodies, including bispecific IgG, show some promise in clinical trials as a means to extend the therapeutic potential of antibodies. Bispecific IgG can be made by separate expression and purification of each parent half antibody followed by in vitro reconstitution. Generating bispecific IgG by coexpression of two different light and heavy chains in a single host cell is potentially more efficient because it obviates the need for two separate cell lines and purification processes. However, this workflow may produce unwanted mispaired IgG species in addition to the desired bispecific IgG. Development and identification of designs that facilitate cognate light chain pairing may benefit from more refined methods to identify and quantify low levels of mispaired IgG. Using an anti-IL-4/IL-13 bispecific IgG, a mass spectrometric characterization method was developed using native or denaturing conditions by direct infusion into an Exactive Plus Extended Mass Range Orbitrap instrument. The high mass resolving power of the instrument allows unambiguous identification and accurate quantification of all light and heavy chain pairing variants in a mixture of bispecific IgG assembled in vivo upon coexpression down to 1% impurity. Preferential pairing of the anti-IL-13 light chain to its cognate heavy chain was observed, which may be leveraged to guide the design of a single-cell solution for streamlined production of bispecific IgG. Additionally, the utility of native mass spectrometry in deconvoluting complex antibody mixtures and in antigen-binding experiments to understand the contribution of doubly light chain mispaired bispecific IgG was demonstrated.
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Luis
authors.initials: L
authors.lastname: Schachner
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Han
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Dillon
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Luke
authors.initials: L
authors.lastname: McCarty
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Diego
authors.initials: D
authors.lastname: Ellerman
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Yiyuan
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Christoph
authors.initials: C
authors.lastname: Spiess
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Jennie R
authors.initials: JR
authors.lastname: Lill
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Carter
authors.affiliation: Departments of Proteomics & Biological Resources, ‡Antibody Engineering, and §Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
mesh_terms: D018033:Antibodies, Bispecific / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018793:Interleukin-13 / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology; D057230:Limit of Detection; D011489:Protein Denaturation; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D018793:Interleukin-13; D015847:Interleukin-4