Enhanced Glycosylation Detection and Characterization of a Human IgG1 Reference Material by using 2D‐LC MS/MS Fractionation and High Resolution Mass Spectral Library
Authors: Dong Qian , Yan Xinjian , Liang Yuxue , Stein Stephen E. 
Published: June 2021

IntroductionThe structure analysis of glycans remains challenging due to the lack of high‐throughput analytical platforms and freely available automated software capable of analyzing spectral data and determine the compositions and structures of glycopeptides and glycans. Here we describe the in‐depth characterization and relative quantification of N‐glycosylation in an IgG1 reference material (NISTmAb) and commercial mAbs using liquid chromatography and tandem mass spectrometer (1D/2D LC‐MS/MS) and the mass spectral library.MethodsDetailed analyses were carried out of glycopeptides produced in the tryptic digestion of the NISTmAb and other commercial mAbs. The method includes (1) nanospray ESI LC–MS/MS over a wide range of HCD collision energies with both conventional 1D separation for various digestion conditions and a 20/24 fraction 2D‐LC study of a single digest; (2) energy‐dependent changes in HCD fragmentation to determine the proposed glycan structures over a wide range of NCE values: 12, 16, 20, 24, 28, 32, 36, and 40; (3) the development of an automated glycopeptide analysis software tool; and (4) the creation of a publicly available high resolution spectral library of glycopeptides. This resulted in 800 1D and 300 2D runs and a glycopeptide spectral library comprising 80 glycans with different masses, and around 2000 HCD spectra of glycopeptides obtained in IgG1 mAbs expressed in NS0 and CHO cell lines.ResultsDerived from 195 different glycopeptides found in 1D and 247 glycopeptides found in 2D runs, more than 60 unique glycan structures in the NISTmAb were confidently determined. This is a significantly larger number of glycans than found in any other glycoanalysis of therapeutic glycoproteins, exhibiting a greater variety of low‐abundance glycan structures detected in 2D studies compared to that of 1D and released glycan studies. This comparison directly illustrates the added depth of the 2D studies leading to the extended abundance range and glycoanalysis coverage. To summarize, the IgG‐Fc N‐linked glycosylation profile of NISTmAb was predominated by asialylated, biantennary core fucosylated complex glycans, which are well‐known main glycoforms observed in IgG1 mAbs produced by all major mammalian cell expression systems. However, significantly higher amounts of α‐linked galactosylation and sialylation were also observed in the 2D study. This profile revealed major differences between the cell lines in the glycoforms produced: the NS0 cell line used for making the IgG1 under study expressed glycoforms with higher extents of nonhuman α‐Gal and Neu5GC residues. In addition, an extensive analysis of HCD glycopeptide fragmentation at multiple low HCD energies confirmed the newly proposed glycan compositions, in‐source glycan fragments, glycan rearrangement products, multiple structural isomers, and glycan modifications including sulfated N‐glycans. This led to the creation of a first high‐resolution, peak‐annotated mass spectral library to aid the analysis of glycopeptides derived from IgG1 drugs. This library is freely available online at http://chemdata.nist.gov/dokuwiki/doku.php?id=peptidew:lib:newigghsalib along with search software.Support or Funding InformationThis work was supported solely with NIST funds. Certain commercial equipment, instruments, or materials are identified in this presentation to specify the experimental procedure adequately. Such an identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.