title: Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins.
abstract: Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.
authors: Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands. r.haselberg@uu.nl
authors: Rob
authors: R
authors: Haselberg
authors: Gerhardus J
authors: GJ
authors: de Jong
authors: Govert W
authors: GW
authors: Somsen
mesh_terms: D000107:Acetylation; D059451:Biosimilar Pharmaceuticals / Q000737:chemistry / Q000378:metabolism*; D003641:Deamination; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D016899:Interferon-beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D036103:Nanotechnology; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013388:Succinimides / Q000737:chemistry
chemical_list: D059451:Biosimilar Pharmaceuticals; D011134:Polysaccharides; D011994:Recombinant Proteins; D013388:Succinimides; C032620:succinimide; D004921:Erythropoietin; D016899:Interferon-beta