Tag: Heparinase

As one of low-molecular-weight heparins (LWMHs), enoxaparin sodium has been manufactured as a generic anticoagulant under different brands, and regulators have been updating requirements. For example, the percentage of polysaccharide chains bearing 1,6-anhydro structure, is a big challenge for enoxaparin manufacturers, according to the test method specified in latest European Pharmacopoeia.

System suitability required by the European Pharmacopoeia Method:

– peak area ratio: maximum 1.15 for the peaks due to 1,6-anhydro ΔIS-IS and 1,6-anhydro ΔIS in the chromatogram obtained with reference solution (b);
– maximum 0.02 for the peaks due to ΔIS and reduced ΔIS in the chromatogram obtained with reference solution (c);
– resolution: minimum 1.5 between the peaks due to reduced ΔIA and 1,6-anhydro ΔIS in the chromatogram obtained with refer-ence solution (c);
– the content of 1,6-anhydro derivatives in enoxaparin sodium CRS is within 1.5 percent of the assigned content.

EP9.0 method 1097

To test the level of 1,6-anhydro species, a combination of heparinase I, heparinase II and heparinase III is used to degrade enoxaparin samples, followed by reduction via sodium tetrahydroborate. The degraded and reduced sample solutions are injected into a HPLC system for analysis.

To meet the system suitability requirements specified by European Pharmacopoeia, exhaustive depolymerization of enoxaparin samples is necessary. To achieve exhaustive depolymerization, enoxaparin manufacturers tend to use native heparinases with high purity and stability.

Through our experience in serving major enoxaparin manufacturers, Asnail has improved further native heparinases, making 1,6-anhydro derivative test for EP 1079 method more robust and confident. Now we lanuch these heparinases in separate SKUs.

• Heparinase I, for EP Method
• Heparinase II, for EP Method
• Heparinase III, for EP Method

To request a quote, please contact Asnail (info@asnailtech.com).

Besides, we are also developing SAX columns for determining 1,6-anhydro derivatives. Initial tests show good column performance, with the two peaks due to 1,6-anhydro ΔIS and 1,6-anhydro ΔIS^epi separated clearly. These SAX columns have been used by customers in China.

The percentage of 1,6-anhydro species is a unique characteristics of enoxaparin sodium, one of low-molecular-weight heparins (LWMHs) that have been widely used as anticoagulants.

How to determine this structure?

Procedures to determine levels of 1,6-anhydro structure are listed in pharmacopeias like EP <1097> and USP <207>.

To determine the percentage of 1,6-anhydro species, enoxaparin sodium samples are first degraded via a combination of heparinases I, II and III, followed by reduction via sodium borohydride. The degraded and reduced sample solutions are injected into a HPLC system for analysis.

Successful analysis of this unique characteristics in enoxaparin sodium heavily depends on the choice of right HPLC columns and heparinases.

Why choose Asnail?

For analysis of 1,6-anhydro species in enoxaparin sodium, Asnail offers heparinases, heparin-derived unsaturated disaccharides, and SAX columns.

Heparinases are the most important and expensive reagents used in QA of enoxaparin sodium. Asnail’s native heparinases feature great stability and purity. By now, Asnail’s heparinases have served many customers whose procedures are in compliance with EN and USP.

To identify peaks in chromatograms, Asnail provides heparin unsaturated disaccharides as reference materials which are prepared by degrading heparin and/or heparan sulfate via heparinases.

For HPLC columns, Asnail has tested and selected the Ultisil XB-SAX analytical column, which has proved to provide better performance than previously available columns. This column is co-developed with Welchmat, a long-time parter of Asnail.

Product links

Heparinases from Flavobacterium heparinum

Heparin-derived unsaturated disaccharides

Ultisil XB-SAX column for 1,6-anhydro test