LMWH is a class of anticoagulant medications used in the prevention of blood clots and treatment of deep vein thrombosis, myocardial infarction and pulmonary embolism. A number of LMWH products are on the market, each similar in structure but created using different initial chemical procedures e.g. Enoxaparin is created using alkaline beta-eliminative cleavage of the benzyl ester of heparin.
Biosimilar Characterization Considerations
These analyses are performed on samples “as received” and are also applicable to Antithrombin III high and low affinity binding fractions of LMWH, providing a more detailed structural assessment of these two sub-populations.
- Intact molecular weight analysis, using on-line LC/ES-MS in negative ion mode and MALDI-TOF MS.
- Strong anion exchange chromatography (SAX), performed on material “as received” and also after gel permeation chromatographic separation into discrete saccharide populations.
- Gel permeation chromatography (GPC) with UV detection at 234nm and Refractive Index (RI) detection.
- Analysis of the products of individual Heparinase digests using SAX. Digestions are performed using Heparinases I, II and III in separate experiments.
- Analysis of the 1,6-Anhydro containing components. Samples are digested with a mixture of Heparinases I, II and III and the products are analysed using SAX.
- 1D and 2D NMR analysis is performed of the samples “as received” and using GPC sub-fractions.
- Secondary and tertiary structure using Circular Dichroism (CD) in the far UV mode.
- Scanning UV spectrophotometry.
Product Specific Technical Considerations
The complexity of the saccharide mixtures within these products (e.g. Degree of Polymerization (DP) and levels of sulphation) means that high end analytics should be applied within characterization and comparability assessments. The methods above have been implemented and developed by BioPharmaSpec to provide a wide breadth of structural and physicochemical data to allow rigorous assessments of LWMH products to be made.