A multi-method approach toward de novo glycan characterization: a Man-5 case study.

Regulatory agencies' expectations for biotherapeutic approval are becoming more stringent with regard to product characterization, where minor species as low as 0.1% of a given profile are typically identified. The mission of this manuscript is to demonstrate a multi-method approach toward de novo glycan characterization and quantitation, including minor species at or approaching the 0.1% benchmark. Recently, unexpected isomers of the Man(5)GlcNAc(2) (M(5)) were reported (Prien JM, Ashline DJ, Lapadula AJ, Zhang H, Reinhold VN. 2009. The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap mass spectrometry (MS). J Am Soc Mass Spectrom. 20:539-556). In the current study, quantitative analysis of these isomers found in commercial M(5) standard demonstrated that they are in low abundance (<1% of the total) and therefore an exemplary "litmus test" for minor species characterization. A simple workflow devised around three core well-established analytical procedures: (1) fluorescence derivatization; (2) online rapid resolution reversed-phase separation coupled with negative-mode sequential mass spectrometry (RRRP-(-)-MS(n)); and (3) permethylation derivatization with nanospray sequential mass spectrometry (NSI-MS(n)) provides comprehensive glycan structural determination. All methods have limitations; however, a multi-method workflow is an at-line stopgap/solution which mitigates each method's individual shortcoming(s) providing greater opportunity for more comprehensive characterization. This manuscript is the first to demonstrate quantitative chromatographic separation of the M(5) isomers and the use of a commercially available stable isotope variant of 2-aminobenzoic acid to detect and chromatographically resolve multiple M(5) isomers in bovine ribonuclease B. With this multi-method approach, we have the capabilities to comprehensively characterize a biotherapeutic's glycan array in a de novo manner, including structural isomers at >/=0.1% of the total chromatographic peak area.

[1]  D. Ashline,et al.  Carbohydrate structural isomers analyzed by sequential mass spectrometry. , 2007, Analytical chemistry.

[2]  C W Sutton,et al.  Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. , 1994, Analytical biochemistry.

[3]  V. Reinhold,et al.  Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole Ion trap mass spectrometer: neutral oligosaccharides and N-linked glycans. , 1998, Analytical chemistry.

[4]  Yehia Mechref,et al.  Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry. , 2003, Analytical chemistry.

[5]  T. Hayakawa,et al.  Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. , 2003, Analytical biochemistry.

[6]  J. Thomas-Oates,et al.  Sodium-cationized oligosaccharides do not appear to undergo 'internal residue loss' rearrangement processes on tandem mass spectrometry. , 1998, Rapid communications in mass spectrometry : RCM.

[7]  G Chataigné,et al.  Polysaccharides analysis of sinorhizobial capside by on-line anion exchange chromatography with pulsed amperometric detection and mass spectrometry coupling. , 2008, Journal of chromatography. A.

[8]  D. Harvey Fragmentation of negative ions from carbohydrates: Part 2. Fragmentation of high-mannose N-linked glycans , 2005, Journal of the American Society for Mass Spectrometry.

[9]  D. Harvey Collision-induced fragmentation of negative ions from N-linked glycans derivatized with 2-aminobenzoic acid. , 2005, Journal of mass spectrometry : JMS.

[10]  Yehia Mechref,et al.  Solid-phase permethylation of glycans for mass spectrometric analysis. , 2005, Rapid communications in mass spectrometry : RCM.

[11]  Yelena Lyubarskaya,et al.  A comparison of three techniques for quantitative carbohydrate analysis used in characterization of therapeutic antibodies. , 2006, Carbohydrate research.

[12]  A. M. Lawson,et al.  Branching pattern and sequence analysis of underivatized oligosaccharides by combined MS/MS of singly and doubly charged molecular ions in negative-ion electrospray mass spectrometry , 2002, Journal of the American Society for Mass Spectrometry.

[13]  André M Deelder,et al.  Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments. , 2006, Rapid communications in mass spectrometry : RCM.

[14]  M. Karas,et al.  Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 2: Application to isomeric mixtures) , 2002, Journal of the American Society for Mass Spectrometry.

[15]  J. Zaia,et al.  The role of mobile protons in negative ion CID of oligosaccharides , 2007, Journal of the American Society for Mass Spectrometry.

[16]  A. M. Lawson,et al.  Negative-ion electrospray mass spectrometry of neutral underivatized oligosaccharides. , 2001, Analytical chemistry.

[17]  B. Reinhold,et al.  Carbohydrate molecular weight profiling, sequence, linkage, and branching data: ES-MS and CID. , 1995, Analytical chemistry.

[18]  Y. C. Lee High-performance anion-exchange chromatography for carbohydrate analysis. , 1990, Analytical biochemistry.

[19]  R. Dwek,et al.  Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry. , 2008, Analytical biochemistry.

[20]  P. Vouros,et al.  Liquid chromatography ion trap mass spectrometric analysis of oligosaccharides using permethylated derivatives. , 2001, Rapid communications in mass spectrometry : RCM.

[21]  D. Harvey Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates. , 2005, Rapid communications in mass spectrometry : RCM.

[22]  J. Cipollo,et al.  A glycomics platform for the analysis of permethylated oligosaccharide alditols , 2007, Journal of the American Society for Mass Spectrometry.

[23]  Justin M. Prien,et al.  The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS , 2009, Journal of the American Society for Mass Spectrometry.

[24]  David J Harvey,et al.  Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates , 2005, Journal of the American Society for Mass Spectrometry.

[25]  B. Domon,et al.  Structural analysis of permethylated oligosaccharides by electrospray tandem mass spectrometry. , 1997, Analytical chemistry.

[26]  D. Harvey Fragmentation of negative ions from carbohydrates: Part 3. Fragmentation of hybrid and complex N-linked glycans , 2005, Journal of the American Society for Mass Spectrometry.

[27]  Niclas G Karlsson,et al.  Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. , 2004, Journal of the American Society for Mass Spectrometry.

[28]  D. Ashline,et al.  Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn. , 2005, Analytical chemistry.

[29]  Gregory C Flynn,et al.  Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry. , 2007, Analytical biochemistry.

[30]  R. O'neill,et al.  A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry. , 1994, Carbohydrate research.

[31]  Philip G. Evans,et al.  Analysis of chain and blood group type and branching pattern of sialylated oligosaccharides by negative ion electrospray tandem mass spectrometry. , 2006, Analytical chemistry.

[32]  Carlito Lebrilla,et al.  Nanoliquid chromatography‐mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high‐accuracy mass analyzer , 2005, Electrophoresis.

[33]  R. Dwek,et al.  "Internal residue loss": rearrangements occurring during the fragmentation of carbohydrates derivatized at the reducing terminus. , 2002, Analytical chemistry.

[34]  J. Rohrer,et al.  Improvements to in-line desalting of oligosaccharides separated by high-pH anion exchange chromatography with pulsed amperometric detection. , 1998, Analytical biochemistry.

[35]  Hildegard Geyer,et al.  Strategies for analysis of glycoprotein glycosylation. , 2006, Biochimica et biophysica acta.

[36]  D. Harvey,et al.  Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry. , 1997, Rapid communications in mass spectrometry : RCM.

[37]  V. Reinhold,et al.  Detailed characterization of carbohydrate linkage and sequence in an ion trap mass spectrometer: glycosphingolipids. , 1998, Analytical biochemistry.

[38]  M. Karas,et al.  Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 1: Methodology) , 2002, Journal of the American Society for Mass Spectrometry.

[39]  A. Deelder,et al.  Normal-phase nanoscale liquid chromatography-mass spectrometry of underivatized oligosaccharides at low-femtomole sensitivity. , 2004, Analytical chemistry.

[40]  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.

[41]  D. Harvey,et al.  Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. , 1999, Mass spectrometry reviews.

[42]  Bradley D Prater,et al.  Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins. , 2007, Analytical biochemistry.

[43]  B. Domon,et al.  Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry. , 1998, Analytical chemistry.

[44]  J. Rosa,et al.  Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans. , 2006, Glycobiology.

[45]  R. Townsend,et al.  Techniques in Glycobiology , 1997 .

[46]  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.

[47]  T. Seyfried,et al.  Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry. , 2008, Glycobiology.

[48]  C. Costello,et al.  Collisionally activated dissociation and electron capture dissociation provide complementary structural information for branched permethylated oligosaccharides , 2008, Journal of the American Society for Mass Spectrometry.

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

[50]  B. Domon,et al.  Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry. , 1988, Biochemistry.

[51]  Y. Mechref,et al.  Electrophoretic analysis of N-glycans on microfluidic devices. , 2007, Analytical chemistry.

[52]  Ionel Ciucanu,et al.  Elimination of oxidative degradation during the per-O-methylation of carbohydrates. , 2003, Journal of the American Chemical Society.

[53]  D. Ashline,et al.  Congruent strategies for carbohydrate sequencing. 3. OSCAR: an algorithm for assigning oligosaccharide topology from MSn data. , 2005, Analytical chemistry.

[54]  Niclas G Karlsson,et al.  Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. , 2004, Rapid communications in mass spectrometry : RCM.

[55]  A. van Dorsselaer,et al.  Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer. , 2004, Rapid communications in mass spectrometry : RCM.

[56]  Qiang Qin,et al.  High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry. , 2009, Analytical biochemistry.

[57]  Gregory C Flynn,et al.  The effect of Fc glycan forms on human IgG2 antibody clearance in humans. , 2008, Glycobiology.

[58]  K. Anumula,et al.  Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. , 2006, Analytical biochemistry.

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

[60]  D. Harvey,et al.  Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides. , 1998, Analytical chemistry.

[61]  Michael Pierce,et al.  Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I. , 2007, Glycobiology.

[62]  C. Lebrilla,et al.  Evidence for long-range glycosyl transfer reactions in the gas phase , 2002, Journal of the American Society for Mass Spectrometry.

[63]  M. Schachner,et al.  Glycomic analysis of N-linked carbohydrate epitopes from CD24 of mouse brain. , 2009, Journal of proteome research.

[64]  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.