Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis

ABSTRACT Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

[1]  Marcella Yu,et al.  Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans , 2012, mAbs.

[2]  Ying Ge,et al.  Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies. , 2018, Analytical chemistry.

[3]  K. Shitara,et al.  The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity* , 2003, The Journal of Biological Chemistry.

[4]  K. Yoshino,et al.  Use of the derivatizing agent 4-aminobenzoic acid 2-(diethylamino)ethyl ester for high-sensitivity detection of oligosaccharides by electrospray ionization mass spectrometry. , 1995, Analytical chemistry.

[5]  L. Presta,et al.  Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity* , 2002, The Journal of Biological Chemistry.

[6]  C. Lebrilla,et al.  Annotation of a serum N-glycan library for rapid identification of structures. , 2012, Journal of proteome research.

[7]  John E. Schiel,et al.  Comparison of Traditional 2-AB Fluorescence LC-MS/MS and Automated LC-MS for the Comparative Glycan Analysis of Monoclonal Antibodies. , 2015, Journal of pharmaceutical sciences.

[8]  Hongwei Xie,et al.  Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies , 2010, mAbs.

[9]  Y. Yu,et al.  Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. , 2015, Analytical chemistry.

[10]  Pauline M. Rudd,et al.  GlycoBase and autoGU: tools for HPLC-based glycan analysis , 2008, Bioinform..

[11]  R. Dwek,et al.  A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. , 1996, Analytical biochemistry.

[12]  S. Nicolardi,et al.  Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry , 2018, Analytical chemistry.

[13]  L. R. Ruhaak,et al.  Glycan labeling strategies and their use in identification and quantification , 2010, Analytical and bioanalytical chemistry.

[14]  J. Stadlmann,et al.  Mass + retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans. , 2007, Analytical chemistry.

[15]  A. Varki,et al.  Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins , 2010, Nature Biotechnology.

[16]  Quynh-Thu Le,et al.  Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. , 2008, The New England journal of medicine.

[17]  Scott R. Kronewitter,et al.  The glycolyzer: Automated glycan annotation software for high performance mass spectrometry and its application to ovarian cancer glycan biomarker discovery , 2012, Proteomics.

[18]  A. Beck.  Biosimilar, biobetter and next generation therapeutic antibodies , 2011, mAbs.

[19]  Pauline M Rudd,et al.  A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes , 2018, Scientific Reports.

[20]  W. Alley,et al.  Advanced assessment of the physicochemical characteristics of Remicade® and Inflectra® by sensitive LC/MS techniques , 2016, mAbs.

[21]  P. Rudd,et al.  Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent. , 2010, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[22]  R. Parekh,et al.  Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. , 1995, Analytical biochemistry.

[23]  Ying Qing Yu,et al.  N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality. , 2014, Analytical chemistry.

[24]  Quynh-Thu Le,et al.  Cetuximab-Induced Anaphylaxis and IgE Specific for Galactose-α-1,3-Galactose , 2008 .

[25]  Z. Shriver,et al.  Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins , 2010, Nature Biotechnology.

[26]  Pauline M. Rudd,et al.  Orthogonal Technologies for NISTmAb N-Glycan Structure Elucidation and Quantitation , 2015 .

[27]  Alain Van Dorsselaer,et al.  Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry. , 2012, Analytical chemistry.

[28]  Yehia Mechref,et al.  High-throughput solid-phase permethylation of glycans prior to mass spectrometry. , 2008, Rapid communications in mass spectrometry : RCM.