title: Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies.
abstract: Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Yang
authors.affiliation: b Computational Biology, Adimab , Palo Alto , CA , USA.
authors.forename: Tushar
authors.initials: T
authors.lastname: Jain
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Lynaugh
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: R Paul
authors.initials: RP
authors.lastname: Nobrega
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Lu
authors.affiliation: b Computational Biology, Adimab , Palo Alto , CA , USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Boland
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Burnina
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Tingwan
authors.initials: T
authors.lastname: Sun
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Caffry
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Brown
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Zhi
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Asparouh
authors.initials: A
authors.lastname: Lilov
authors.affiliation: a Protein Analytics, Adimab , Lebanon , NH , USA.
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction
chemical_list: D000911:Antibodies, Monoclonal; D008715:Methionine; D006861:Hydrogen Peroxide
keywords: Chemical liability; chemical stability; developability; forced degradation; mAbs; methionine; oxidation; solvent accessible surface area