, liquid chromatography coupled with tandem mass spectrometry; mAb, monoclonal antibody; MS 1 , a mass to charge ratio survey scan; MS 2 , tandem mass spectrometry -selected ions from MS 1 are fragmented and fragment ion mass measured; UPLC, ultrahigh performance liquid chromatography 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.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion (huLCAT-Fc) is a chimeric protein produced by fusing human Fc to the C-terminus of the human enzyme via a linker sequence. The huLCAT-Fc homodimer contains five N-linked glycosylation sites per monomer. The heterogeneity and site-specific distribution of the various glycans were examined using enzymatic digestion and LC-MS=MS, followed by automatic processing. Almost all of the N-linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N-linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N-linked site exclusively contain typical asialobiantennary structures. HuLCAT-Fc was also confirmed to have mucin-type glycans attached at T 407 and S 409 . When LCAT-Fc fusions were constructed using a G-S-G-G-G-G linker, an unexpected 1632 Da xylosebased glycosaminoglycan (GAG) tetrasaccharide core of Xyl-Gal-Gal-GlcA was attached to S 418 . Several minor intermediate species including Xyl, Xyl-Gal, Xyl-Gal-Gal, and a phosphorylated GAG core were also present. The mucin-type O-linked glycans can be effectively released by sialidase and O-glycanase; however, the GAG could only be removed and localized using chemical alkaline b-elimination and targeted LC-MS=MS. E 416 (the C-terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O-xylosyltransferase. HuLCAT-Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPPE 416 GS 418 GGGGDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.