A simplified non-reduced peptide mapping method with faster and efficient enzymatic digestion for characterization of native disulfide bonds in monoclonal and bispecific antibodies.

Autor: Gu L; Biologics Analytical Science, Incyte Corporation, 1801 Augustine Cut-off, Wilmington, DE 19803, USA. Electronic address: lgu@incyte.com., Hu TX; Biologics Analytical Science, Incyte Corporation, 1801 Augustine Cut-off, Wilmington, DE 19803, USA.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2024 Nov 15; Vol. 250, pp. 116400. Date of Electronic Publication: 2024 Aug 06.
DOI: 10.1016/j.jpba.2024.116400
Abstrakt: Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liqing Gu reports financial support was provided by Incyte Corporation. Tiger X. Hu reports financial support was provided by Incyte Corporation. Liqing Gu reports a relationship with Incyte Corporation that includes: employment and equity or stocks. Tiger X. Hu reports a relationship with Incyte Corporation that includes: employment and equity or stocks.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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