Dataset Information

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

ABSTRACT: Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement.

SUBMITTER: Sanderson P

PROVIDER: S-EPMC5862776 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

Sanderson Patience P Stickney Morgan M Leach Franklin E FE Xia Qiangwei Q Yu Yanlei Y Zhang Fuming F Linhardt Robert J RJ Amster I Jonathan IJ

Journal of chromatography. A 20180224

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been deve ...[more]

PMID: 29501428

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Project description:Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins.

Project description:Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.

Dataset Information

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

Publications

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

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