title: Absolute Quantitation of Intact Recombinant Antibody Product Variants Using Mass Spectrometry.
abstract: Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Because of the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and noncovalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of noncovalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequences with their corresponding halves and bsAb, it is not suitable to use peptides as surrogates for their quantitation. To tackle these analytical challenges, we developed a mass spectrometry-based quantitation method. Chip-based nanoflow LC-time-of-flight mass spectrometry coupled with a standard addition approach provided unbiased absolute quantitation of these drug-product-related variants. Two methods for the addition of known levels of standard (multi- or single-addition) were evaluated. Both methods demonstrated accurate and reproducible quantitation of homodimers at the 0.2% (w/w) level, with the single-addition method having the promise of higher analytical throughput.
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Frank D
authors.initials: FD
authors.lastname: Macchi
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Charlene
authors.initials: C
authors.lastname: Li
authors.affiliation: Eurofins Lancaster Laboratories, Inc. , 2425 New Holland Pike, Lancaster, Pennsylvania 17601, United States.
authors.forename: Chenchen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Anh Nguyen
authors.initials: AN
authors.lastname: Dang
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Marhoul
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Hui-min
authors.initials: HM
authors.lastname: Zhang
authors.affiliation: Purification Development, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Tully
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Liu
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: X Christopher
authors.initials: XC
authors.lastname: Yu
authors.affiliation: Protein Analytical Chemistry, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Michels
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry*; D016466:CHO Cells; D003412:Cricetulus; D013058:Mass Spectrometry / Q000295:instrumentation; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins