Mishra, S.
, Kaur, A.
and Bhavaraju, S.
(2026),
Comprehensive solution NMR characterization of the direct thrombin inhibitor peptide bivalirudin and its process impurities by heteronuclear mapping at natural isotopic abundance., Journal of Pharmaceutical and Biomedical Analysis, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=960005 (Accessed August 14, 2026)
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Comprehensive solution NMR characterization of the direct thrombin inhibitor peptide bivalirudin and its process impurities by heteronuclear mapping at natural isotopic abundance.
Published
Author(s)
, Anupreet Kaur, Sitaram Bhavaraju
Abstract
Bivalirudin is a 20-residue synthetic peptide that functions as a bi-valent thrombin inhibitor by simultaneously engaging thrombin's active site and exosite I. Despite its widespread clinical use, comprehensive NMR-based characterization of bivalirudin and its process-related impurities has not been reported. Here, we present the first complete heteronuclear NMR analysis of bivalirudin and two major impurities—Asp9-bivalirudin (a deamidation product) and desGlu13-bivalirudin (a synthesis deletion)—at natural isotopic abundance. Using a combination of 1D and 2D 1H, 13C, and 15N NMR experiments in DMSO-d₆ and aqueous solution, we achieved near-complete backbone and side-chain assignments, now deposited in the BMRB. We demonstrate that 2D heteronuclear amide maps can unambiguously differentiate the parent peptide from its variants. Additionally, we report the first solution-state NMR structure of free bivalirudin, revealing a compact architecture wherein the active site– and exosite-binding segments are spatially proximate, potentially reflecting a pre-binding intermediate. This study establishes a durable spectral reference framework and simplified 2D heteronuclear spectral fingerprints for structural integrity assessment and impurity tracking of bivalirudin.Citation
Journal of Pharmaceutical and Biomedical Analysis
Volume
281
Pub Type
Journals
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Keywords
bivalirudin, Asp9-bivalirudin, desGlu13-bivalirudin, peptide impurities, NMR, natural abundance heteronuclear NMR, peptide synthesis impurities
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Created July 29, 2026, Updated August 13, 2026