title: A chemical and computational approach to comprehensive glycation characterization on antibodies.
abstract: Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS(2) mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS(1) based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.
authors.affiliation: a Drug Substance Development; Amgen Inc. ; Seattle , WA , USA.
authors.forename: Ramsey A
authors.initials: RA
authors.lastname: Saleem
authors.forename: Brittany R
authors.initials: BR
authors.lastname: Affholter
authors.forename: Sihong
authors.initials: S
authors.lastname: Deng
authors.forename: Phil C
authors.initials: PC
authors.lastname: Campbell
authors.forename: Kelli
authors.initials: K
authors.lastname: Matthies
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Eakin
authors.forename: Alison
authors.initials: A
authors.lastname: Wallace
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D007074:Immunoglobulin G
keywords: BA, boronate affinity chromatography; CDR3, complementary-determining region 3; CEX, cation exchange chromatography; CID, collision induced dissociation; CV, coefficient of variation; Da, daltons; EIC, extracted ion chromatogram; HC-CDR3, heavy chain complementary determining region 3; HPLC, high performance liquid chromatography; LC-MS2, liquid chromatography coupled with tandem mass spectrometry; MS1, a mass to charge ratio survey scan; MS2, tandem mass spectrometry - selected ions from MS1 are fragmented and fragment ion mass measured; UPLC, ultrahigh performance liquid chromatography; boronate affinity chromatography; glycation; mAb, monoclonal antibody; structural modeling; targeted mass spectrometry; Å, angstroms