Category: News

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.

Asnail now offers SEC columns for molecular weight determinations that can reduce cost per injection to 1/4 or lower, compared to columns from other vendors.

Molecular weight (MW) and its distribution are important characteristics of heparin and low molecular weight heparins (LMWHs), a family of anticoagulants with polydisperse, heterogeneous polysaccharide chains.

Current compendial methods for heparin molecular weight determinations involve separation on size-exclusion chromatography (SEC) columns, followed by detection with refractive index detectors (RID). The resulting calibration curve relating log10(MW) to retention time can be obtained by 3rd order polynominal fitting.

While some SEC columns have been widely used in heparin industry before, they are subject to short life time. BioCore SEC-HP columns, a series of SEC columns tailored for heparin MW determinations, are used for MW determinations of heparin and LMWHs. The results show good performance which meets the system suitability required by the latest methods specified in pharmacopoeias. Besides, these columns show very long life time, without performance loss even after 100 injections, which can reduce cost per injection for heparin industry.

Product links

BioCore SEC-150HP Columns for Low-Molecular-Weight Heparins

BioCore SEC-300HP & SEC-500HP Columns for Unfractionated Heparins

Related documents

Asnail SEC Columns Catalog

Molecular Weight Determinations of Heparin and Low-Molecular-Weight Heparins

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

Asnail now offers chromogenic substrates (S-2222 and S-2765) for factor VIII assay. The chromogenic assay is recommended for clinical diagnosis of haemophilia, monitoring factor replacement therapies and potency assignment of recombinant factor VIII.

Coagulation factor VIII (FVIII) performs a critical role in the intrinsic blood coagulation pathway. The typical quantitative chromogenic assay for coagulation factor VIII is specified in European Pharmacopoeia, as a two-stage procedure.

In this assay, coagulation factor VIII first catalyzes the transformation of factor X into its activated form, factor Xa, which in turn catalyzes the release of pNA from the substrate (S-2222 or S-2765). The potency of coagulation factor VIII is deduced by measuring the chromophore, pNA.

Asnail now offers two chromogenic substrates for this assay.

• S-2222, Chromogenic Substrate for Factor Xa, for Coagulation Testing
• S-2765, Chromogenic Substrate for Factor Xa, for Coagulation Testing

Asnail now offers chromogenic substrates in gram grade for coagulation testing.

These large-quantity chromogenic substrates can be used in chromogenic assay kits.

• S-2222, Chromogenic Substrate for Factor Xa, for Coagulation Testing
• S-2765, Chromogenic Substrate for Factor Xa, for Coagulation Testing
• S-2238, Chromogenic Substrate for Thrombin, for Coagulation Testing

For more information, please contact us via info@asnailtech.com.

Asnail now offers a wide range of reagents used in chromogenic assays for heparin.

Chromogenic Substrates

Asnail’s chromogenic substrates including:

S-2238, Chromogenic Substrate for Thrombin

S-2222, Chromogenic Substrate for Factor Xa

S-2765, Chromogenic Substrate for Factor Xa

Coagulation Factors

Asnail’s coagulation factors including:

Activated Factor X (FXa)

Alpha-Thrombin (FIIa)

Antithrombin

Asnail provides human antithrombin for both anti-FIIa and anti-FXa assays.

Human Antithrombin

With our expertise in heparin analysis, we also offer technical support with regard to chromogenic assays for heparin and LMWHs.

For more information, please contact us.

Asnail provides heparinases for heparin analysis, particularly for determining the 1,6-anhydro derivatives in exnoxaparin sodium.

All these heparinases are native enzymes from from Flavobacterium Heparinum. Please check the following links for detailed information.

Heparinase I

Heparinase II

Heparinase III

Click here to download the heparinases catalog.

For more information, please contact Asnail.