title: Complementary MS methods assist conformational characterization of antibodies with altered S-S bonding networks.
abstract: As therapeutic monoclonal antibodies (mAbs) become a major focus in biotechnology and a source of the next-generation drugs, new analytical methods or combination methods are needed for monitoring changes in higher order structure and effects of post-translational modifications. The complexity of these molecules and their vulnerability to structural change provide a serious challenge. We describe here the use of complementary mass spectrometry methods that not only characterize mutant mAbs but also may provide a general framework for characterizing higher order structure of other protein therapeutics and biosimilars. To frame the challenge, we selected members of the IgG2 subclass that have distinct disulfide isomeric structures as a model to evaluate an overall approach that uses ion mobility, top-down MS sequencing, and protein footprinting in the form of fast photochemical oxidation of proteins (FPOP). These three methods are rapid, sensitive, respond to subtle changes in conformation of Cys → Ser mutants of an IgG2, each representing a single disulfide isoform, and may be used in series to probe higher order structure. The outcome suggests that this approach of using various methods in combination can assist the development and quality control of protein therapeutics.
authors.affiliation: Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Jones
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Weidong
authors.initials: W
authors.lastname: Cui
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Justin B
authors.initials: JB
authors.lastname: Sperry
authors.forename: James A
authors.initials: JA
authors.lastname: Carroll
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D011994:Recombinant Proteins