High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

Publisher: Wiley-VCH Country of Publication: Germany NLM ID: 101088554 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1615-9314 (Electronic) Linking ISSN: 16159306 NLM ISO Abbreviation: J Sep Sci Subsets: MEDLINE

Original Publication: Weinheim, Germany : Wiley-VCH, c2001-

Antibodies, Monoclonal*/chemistry ; Electrophoresis, Capillary*/methods; Chromatography, Liquid ; Glycosylation ; Mass Spectrometry/methods

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.
(© 2022 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)

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FKZ 13FH635IB6 Bundesministerium für Bildung und Forschung

Keywords: antibodies; glycosylation; high throughput screening; mass spectrometry

0 (Antibodies, Monoclonal)

Date Created: 20220119 Date Completed: 20220629 Latest Revision: 20220629

20240829

10.1002/jssc.202100865

35044720