Top-down and bottom-up analysis of commercial enoxaparins

- Four types of commercial enoxaparins were analyzed by integrated LC-MS and NMR analysis.
- Heparinase treatment was used before bottom-up analysis and disaccharide analysis.
- Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS.
- Monosaccharide compositional analysis relied on top-down NMR analysis.
- Generic enoxaparins and innovator product are similar, differences are observed due to parent heparin and process conditions.

A strategy for the comprehensive analysis of low molecular weight (LMW) heparins is described that relies on using an integrated top-down and bottom-up approach. Liquid chromatography-mass spectrometry, an essential component of this approach, is rapid, robust, and amenable to automated processing and interpretation. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues comprising a low molecular weight heparin. Using our integrated approach four different low molecular weight heparins prepared from porcine heparin through chemical β-eliminative cleavage were comprehensively analyzed. Lovenox™ and Clexane™, the innovator versions of enoxaparin marketed in the US and Europe, respectively, and two generic enoxaparins, from Sandoz and Teva, were analyzed. The results which were supported by analysis of variation (ANOVA), while showing remarkable similarities between different versions of the product and good lot-to-lot consistency of each product, also detects subtle differences that may result from differences in their manufacturing processes or differences in the source (or parent) porcine heparin from which each product is prepared.