A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format.

Autor:	Ortega-Rodriguez U; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Bettinger JQ; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Zou G; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Falkowski VM; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Lehtimaki M; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Matthews AM; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Biel TG; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Pritts JD; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Wu WW; Facility for Biotechnology Resources, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Shen RF; Facility for Biotechnology Resources, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Agarabi C; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Rao VA; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Xie H; Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA., Ju T; Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA. Electronic address: tongzhong.ju@fda.hhs.gov.
Jazyk:	angličtina
Zdroj:	Cell reports methods [Cell Rep Methods] 2024 Aug 19; Vol. 4 (8), pp. 100834. Date of Electronic Publication: 2024 Aug 07.
DOI:	10.1016/j.crmeth.2024.100834
Abstrakt:	Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins. Competing Interests: Declaration of interests The authors declare no competing interests. (Published by Elsevier Inc.)
Databáze:	MEDLINE
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