title: Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites.
abstract: Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany.
authors: Uwe
authors: U
authors: Möginger
authors: Bruker, Bremen, Germany.
authors: Anja
authors: A
authors: Resemann
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany.
authors: Christopher E
authors: CE
authors: Martin
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.
authors: Sharavathi
authors: S
authors: Parameswarappa
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.
authors: Subramanian
authors: S
authors: Govindan
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany.
authors: Eike-Christian
authors: EC
authors: Wamhoff
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany.
authors: Felix
authors: F
authors: Broecker
authors: Bruker, Bremen, Germany.
authors: Detlev
authors: D
authors: Suckau
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.
authors: Claney Lebev
authors: CL
authors: Pereira
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.
authors: Chakkumkal
authors: C
authors: Anish
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany.
authors: Peter H
authors: PH
authors: Seeberger
authors: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany.
authors: Daniel
authors: D
authors: Kolarich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002853:Chromatography, Liquid; D000939:Epitopes / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018074:Vaccines, Conjugate / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D011134:Polysaccharides; D018074:Vaccines, Conjugate; C028581:CRM197 (non-toxic variant of diphtheria toxin)