Enoxaparin Sodium – Spectroscopic Fingerprint by 2D NMR
The FDA issued a guidance document in February 2016 entitled “Immunogenicity – Related Considerations for Low Molecular Weight Heparin“. The publication contains non-binding recommendations on how characterize low molecular weight heparins (LMWH), such as Enoxaparin. NMR analysis factors prominently into Criteria 1 and 3 of the guidance document providing additional proof of chemical composition and structure of LMWH. By collecting 2D NMR datasets on the Reference Listed Drug (RLD) and for example, a new production lot of drug, the data can be compared qualitatively and quantitatively for similarity. This “spectroscopic fingerprint” approach is finding increasing use and application in the development, production, and regulatory filing of biosimilars.
The figure below is an expansion of the 2D 1H-13C HSQC NMR spectrum of Enoxaparin Sodium – USP-NF where selected resonances for monosaccharides are shown. Whereas in a 1-dimensional NMR spectrum the area of a peak can be used to quantitate a species, the volume of cross peak represents a quantitative amount in a 2-dimensional NMR spectrum. 2D NMR data allow for more detailed structural analysis of complex molecules, such as LMWHs, by separating potentially overlapping signals in another spectroscopic dimension.
Relative quantitation of key structural features of Enoxaparin mentioned in the non-binding recommendations of the guidance document can be easily performed and reported for process research or filing purposes. Specific examples of the epimerization state of the uronates (Iduronic to Glucuronic Ratio of 75:25) in the repeating disaccharides and the structural nature of the non-reducing end monosaccharides (sulfated to non-sulfated Δ4,5 Uronate at non-reducing end ratio of 95: 5) are shown above. The values obtained by Novatia compare well with literature values of Enoxparin for an Iduronic to Glucuronic ratio of 74:25 and sulfated to non-sulfated Δ4,5 Uronate at non-reducing end ratio of 95:5 (M. Guerrini, S. Guglieri, A. Naggi, R. Sasisekharan and G. Torri, Semin. Thromb. Hemostasis, 2007, 33, 478–487).
Novatia is uniquely positioned to assist research organizations in process development and NMR characterization for regulatory filing of Enoxaparin API and other Low Molecular Weight Heparins (LMWH).