Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.

[1]  André M Deelder,et al.  IgG glycosylation analysis , 2009, Proteomics.

[2]  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.

[3]  R. Contreras,et al.  Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. , 2001, Glycobiology.

[4]  Pauline M Rudd,et al.  Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation. , 2010, Analytical chemistry.

[5]  Erdmann Rapp,et al.  Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. , 2010, Journal of proteome research.

[6]  T. Raju,et al.  Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. , 2000, Glycobiology.

[7]  P. Scolnik.  mAbs , 2009 .

[8]  Andrew C. R. Martin,et al.  Accessing the Kabat antibody sequence database by computer , 1996, Proteins.

[9]  S. Jacobsson,et al.  Development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[10]  A. Guttman,et al.  High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate. , 1996, Analytical biochemistry.

[11]  D. Dimitrov,et al.  Therapeutic Antibodies: Current State and Future Trends – Is a Paradigm Change Coming Soon? , 2008, Methods in molecular biology.

[12]  L. McDonnell,et al.  Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry. , 2010, Analytical chemistry.

[13]  A K Patel,et al.  The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. , 1995, Molecular immunology.

[14]  J. Bones,et al.  Unraveling the glyco-puzzle: glycan structure identification by capillary electrophoresis. , 2013, Analytical chemistry.

[15]  R. Townsend,et al.  Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Guttman,et al.  Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species. , 2014, Carbohydrate research.

[17]  J. Ravetch,et al.  Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation , 2006, Science.

[18]  O. Salas-Solano,et al.  On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies. , 2008, Analytical chemistry.

[19]  M. Wuhrer,et al.  High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer , 2014, mAbs.

[20]  R. Jefferis.  Glycosylation of antibody therapeutics: optimisation for purpose. , 2009, Methods in molecular biology.

[21]  Michael Butler,et al.  Expression systems for therapeutic glycoprotein production. , 2009, Current opinion in biotechnology.

[22]  Cleo Kontoravdi,et al.  Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns , 2010, Biotechnology progress.

[23]  R. Contreras,et al.  Noninvasive diagnosis of liver cirrhosis using DNA sequencer–based total serum protein glycomics , 2004, Nature Network Boston.

[24]  A. Deelder,et al.  High-throughput work flow for IgG Fc-glycosylation analysis of biotechnological samples. , 2013, Analytical biochemistry.

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

[26]  A. Varki,et al.  Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. , 2008, Glycobiology.

[27]  J. Ravetch,et al.  Intravenous gammaglobulin suppresses inflammation through a novel TH2 pathway , 2011, Nature.

[28]  A. Deelder,et al.  Analysis of urinary oligosaccharides in lysosomal storage disorders by capillary high-performance anion-exchange chromatography–mass spectrometry , 2012, Analytical and Bioanalytical Chemistry.

[29]  R. Contreras,et al.  Total serum protein N‐glycome profiling on a capillary electrophoresis‐microfluidics platform , 2004, Electrophoresis.

[30]  A. Deelder,et al.  Protein glycosylation analyzed by normal-phase nano-liquid chromatography--mass spectrometry of glycopeptides. , 2005, Analytical chemistry.

[31]  J. Prestegard,et al.  NMR Analysis Demonstrates Immunoglobulin G N-glycans are Accessible and Dynamic , 2011, Nature chemical biology.

[32]  Zhongqi Zhang,et al.  LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins , 2014, Journal of The American Society for Mass Spectrometry.

[33]  R. Dwek,et al.  Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. , 1997, Biochemistry.

[34]  James E. Bailey,et al.  Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity , 1999, Nature Biotechnology.

[35]  E. Rapp,et al.  Capillary electrophoresis/mass spectrometry of APTS-labeled glycans for the identification of unknown glycan species in capillary electrophoresis/laser-induced fluorescence systems. , 2013, Analytical chemistry.

[36]  A. Jones,et al.  The use of high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor and identify oligosaccharide degradation. , 1996, Analytical biochemistry.

[37]  T. Wurch,et al.  Editorial [Hot Topic: Therapeutic Antibodies and Derivatives: From the Bench to the Clinic(Guest Editor: Alain Beck)] , 2008 .

[38]  M. J. Bailey,et al.  Identification and quantification of N-linked oligosaccharides released from glycoproteins: an inter-laboratory study. , 2008, Glycobiology.

[39]  Ashley C. Gucinski,et al.  Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. , 2013, The Analyst.

[40]  Haixu Tang,et al.  Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012* , 2013, Molecular & Cellular Proteomics.

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

[42]  S. Cavalli,et al.  Analysis of carbohydrates by anion exchange chromatography and mass spectrometry. , 2005, Journal of chromatography. A.

[43]  Lihua Huang,et al.  Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization. , 2006, Analytical biochemistry.

[44]  K R Anumula,et al.  High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. , 1998, Glycobiology.

[45]  David J. Harvey,et al.  HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. , 2008, Analytical biochemistry.

[46]  Erdmann Rapp,et al.  N‐glycan analysis by CGE–LIF: Profiling influenza A virus hemagglutinin N‐glycosylation during vaccine production , 2008, Electrophoresis.

[47]  Roy Jefferis,et al.  Glycosylation as a strategy to improve antibody-based therapeutics , 2009, Nature Reviews Drug Discovery.

[48]  C. Neusüss,et al.  Alignment of laser‐induced fluorescence and mass spectrometric detection traces using electrophoretic mobility scaling in CE‐LIF‐MS of labeled N‐glycans , 2012, Electrophoresis.

[49]  R. Jefferis,et al.  A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins. , 1990, The Biochemical journal.

[50]  M. Moscarello,et al.  Branch specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc beta 1,2-Man alpha 1,3-branch of a complex biantennary Asn-linked oligosaccharide. , 1984, The Journal of biological chemistry.

[51]  Pauline M Rudd,et al.  Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions. , 2006, Methods in molecular biology.

[52]  David Ouellette,et al.  Increased serum clearance of oligomannose species present on a human IgG1 molecule , 2012, mAbs.

[53]  I. Rudan,et al.  Comparative Performance of Four Methods for High-throughput Glycosylation Analysis of Immunoglobulin G in Genetic and Epidemiological Research , 2014, Molecular & Cellular Proteomics.

[54]  A. Lim,et al.  Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer. , 2008, Analytical biochemistry.

[55]  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.

[56]  R. Dwek,et al.  Rapid, sensitive sequencing of oligosaccharides from glycoproteins. , 1997, Current opinion in biotechnology.

[57]  V. Quarmby,et al.  Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities , 2012, mAbs.

[58]  P. Bondarenko,et al.  Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs , 2008, Journal of the American Society for Mass Spectrometry.

[59]  B. Küster,et al.  Comparison of fragmentation modes for the structural determination of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry. , 1995, Rapid communications in mass spectrometry : RCM.

[60]  Zhongqi Zhang,et al.  Mass spectrometry for structural characterization of therapeutic antibodies. , 2009, Mass spectrometry reviews.

[61]  R. Townsend,et al.  High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection. , 1988, Analytical biochemistry.

[62]  Martin Pabst,et al.  Glycan analysis by modern instrumental methods , 2011, Proteomics.

[63]  P. Bondarenko,et al.  High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. , 2011, Glycobiology.

[64]  Pauline M Rudd,et al.  A systematic approach to protein glycosylation analysis: a path through the maze. , 2010, Nature chemical biology.

[65]  R. Dwek,et al.  Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG , 1985, Nature.

[66]  T. Pritchett,et al.  Capillary gel electrophoresis separation of high‐mannose type oligosaccharides derivatized by 1‐aminopyrene‐3,6,8‐trisulfonic acid , 1995, Electrophoresis.

[67]  J. Rohrer,et al.  Separation of neutral asparagine-linked oligosaccharides by high-pH anion-exchange chromatography with pulsed amperometric detection. , 1995, Analytical biochemistry.

[68]  Huijuan Li,et al.  Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry , 2013, mAbs.

[69]  J. Roy.  Glycosylation of antibody therapeutics: optimisation for purpose. , 2009 .

[70]  Nicolle H. Packer,et al.  Comparison of Methods for Profiling O-Glycosylation , 2009, Molecular & Cellular Proteomics.

[71]  A. Deelder,et al.  Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[72]  T. Wurch,et al.  Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. , 2008, Current pharmaceutical biotechnology.

[73]  T. Wurch,et al.  Therapeutic antibodies and derivatives: from the bench to the clinic. , 2008, Current pharmaceutical biotechnology.