Commercial heparin characterised by 2D NMR

Commercial heparin manufactured by different processes has been characterised by 2D NMR spectroscopy to establish the compositions and degree of variability across US manufacturers. The work was carried out by scientists from the FDA who are constantly looking for new procedures to analyse biopharmaceuticals of this type.

They tested seven lots of heparin sodium produced by six manufacturers plus the standard from the US Pharmacopeia. HepNa has a complicated structure, consisting of a chain of up to 35 pairs of disaccharides with a glucosamine residue. The researchers also examined ten lots of a version known as low-molecular-weight heparin (LMWH) from three manufacturers. They are prepared from heparin by different methods that involve partial depolymerisation with nitrous acid, enzymatic β-elimination or chemical β-elimination to give dalteparin, tinzaparin and enoxaparin, respectively.

Using 2D NMR with ¹H-¹³C-heteronuclear single quantum coherence (HSQC), the spectral differences were used to differentiate the species and to examine variations between manufacturers as described in Analytical Methods. For instance, the signals from the anhydromannitol group distinguished dalteparin from HepNa. Tinzaparin differed again by having an unsaturated sulphated uronic acid group but no anhydromannitol. The data allowed the proportions of monosaccharide or disaccharide to be calculated for each heparin.

Each run took up to 11 hours on an instrument with a room-temperature probe for HepNA and 6 hours for LMWH. These times could be reduced with a more modern instrument and a cooled probe to just 3 hours for HepNa.

"The data presented in this work illustrates that NMR tests can be valuable for pharmacopeia drug specification or quality assessment purposes because the process by which the heparin types are made imparts quantifiable fingerprints that can be used to identify critical quality attributes in these complex drugs," the team concluded.