Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin

Abstract

Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin. Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as 'enoxaparin', 'mass spectrometry', 'low molecular weight heparin', 'structural characterization', etc. Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.

Keywords: Low molecular weight heparin; enoxaparin; mass spectrometry; size exclusion chromatography; sulfolyltransferase.

Figure 1:

The schema here describes various parameters that need to be evaluated using various instrumentation approaches to validate LMWH. Also, shown herein are different tests performed for method validation. These aspects are covered for evaluating sameness for any biosimilar or generic molecule.

Figure 2:

These structures here illustrate two of the tetrasaccharide moieties obtained for Enoxaparin. Particularly, here 2 variants with or without 4,5 unsaturation on the non-reducing terminus of enoxaparin is exhibited. Such highly sulfated structures introduce heterogeniety in characterization.

Figure 3:

This schema lists products formed in the presence of specific sulfolyltransferases and specific reactant.

Figure 4:

The LC-MS based fragmentation pattern for a 1,6 anhydro terminated enoxaparin grade. This fragmentation profile illustrates different m/z obtained on cleaving enoxaparin into disaccharides, trisaccharides and tetrasaccharides.