title: A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide.
abstract: A robust, high-throughput method using hydrophilic interaction liquid chromatography (HILIC) coupled with a charged aerosol detector (CAD) is reported as a novel approach for trisulfide quantitation in monoclonal antibodies (mAbs). The products of mAb reduction using tris(2-carboxyethyl)phosphine (TCEP) include a species (TCEP(S)) that is stoichiometrically produced from trisulfides. The TCEP reaction products are chromatographically separated, detected, and quantified by the HILICCAD method. The method was qualified to quantify trisulfides across a range of 1-40% (mol trisulfide/mol mAb). In all tested matrix components, assay linearity and intermediate precision were established with correlation coefficients (R(2))>0.99, and relative standard deviations (RSD)<10%. A method comparability study was performed using peptide mapping LC-MS as an orthogonal measurement. For the range of 1-40% trisulfides, the analysis demonstrates that, on average, HILICCAD reads between 0.95 and 1.10 times the value of LC-MS with 95% confidence. Applications of the HILICCAD method include trisulfide determination in purified mAbs to be used in the production of cysteine-linked antibody-drug conjugates, and in cell culture development studies to understand sources of, and strategies for control of, trisulfides.
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA 94080-4990, United States. Electronic address: christrc@gene.com.
authors.forename: Christopher
authors.initials: C
authors.lastname: Cornell
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA 94080-4990, United States.
authors.forename: Amish
authors.initials: A
authors.lastname: Karanjit
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA 94080-4990, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA way, South San Francisco, CA 94080-4990, United States.
authors.forename: Fredric
authors.initials: F
authors.lastname: Jacobson
mesh_terms: D000336:Aerosols; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry*; D013440:Sulfides / Q000032:analysis*
chemical_list: D000336:Aerosols; D000911:Antibodies, Monoclonal; D003545:Cysteine; D007074:Immunoglobulin G; D010720:Phosphines; D013440:Sulfides; C080938:tris(2-carboxyethyl)phosphine
keywords: Antibody-drug conjugates; Charged aerosol detector; Hydrophilic interaction chromatography; Monoclonal antibodies; Tris(2-carboxyethyl)phosphine (TCEP); Tris(2-carboxyethyl)phosphine-oxide (TCEP[O]); Tris(2-carboxyethyl)phosphine-sulfide (TCEP[S]); Trisulfides