Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Haselberg, Rob; de Jong, Gerhardus J.; Somsen, Govert W.

doi:10.1021/ac303158f

Cited by 149Mass Spectrometry Techniques and ApplicationsGlycosylation and Glycoproteins ResearchMicrofluidic and Capillary Electrophoresis Applications

Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Haselberg, Rob, de Jong, Gerhardus J., Somsen, Govert W.·2013·Analytical Chemistry

DOI 10.1021/ac303158f

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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.

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CE-MS analysis of intact glycoforms estimated glycoform concentrations between 0.35 and 630 nM based on relative intensities.
“Quantitative information was based on the relative intensities of the detected glycoforms where glycoform concentrations were estimated to range between 0.35 to 630 nM.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [134]

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Authors: Haselberg, R., de Jong, G. J., & Somsen, G. W.
Affiliations: Utrecht University (NL)
Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
Corresponding author: Haselberg, Rob
ORCID: Haselberg, Rob
Somsen, Govert W.
Venue: Analytical Chemistry
Publisher: American Chemical Society (ACS)
Published: 2013-01-30
Volume: 85
Issue: 4
Pages: 2289-2296
ISSN: 0003-2700 (ISSN-L)
1520-6882
DOI: 10.1021/ac303158f
OpenAlex: W2318027074
Cited by: 149
References: 45
Citations by year: 2026: 5 · 2025: 6 · 2024: 5 · 2023: 2 · 2022: 10 · 2021: 9 · 2020: 7 · 2019: 16 · 2018: 16 · 2017: 16 · 2016: 16 · 2015: 16 · 2014: 14 · 2013: 11
FWCI: 8.54
Type: journal article
Language: en
Open access: closed
Locations: Analytical Chemistry (publishedVersion)
PubMed (publishedVersion)
VU Research Portal (publishedVersion)
Data Archiving and Networked Services (DANS) (submittedVersion)
Funders: Stichting voor de Technische Wetenschappen
Topics: Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
Keywords: capillary electrophoresis-mass spectrometry, interferon-beta, erythropoietin, ionization efficiency, ion suppression, deamidation, oxidation, acetylation, biosimilars, biopharmaceutical quality control
MeSH terms: Acetylation, Deamination, Erythropoietin, Glycosylation, Humans, Oxidation-Reduction, Polysaccharides, Recombinant Proteins, Succinimides, Interferon-beta, Electrophoresis, Capillary, Spectrometry, Mass, Electrospray Ionization, Nanotechnology, Biosimilar Pharmaceuticals

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Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

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