The Use of ITC and the Software AFFINImeter for the Quantification of the Anticoagulant Pentasaccharide in Low Molecular Weight Heparin
In this chapter, we describe an original protocol based on ITC experiments and data analysis with the software AFFINImeter to get information of heparin-AT interactions relevant for the elucidation of the anticoagulant activity of heparins. This protocol is used to confirm the presence of the bioactive pentasaccharide with anticoagulant activity in heparins and to determine the amount of this pentasaccharide in the sample. Here we have applied this protocol to the characterization of low molecular weight heparins.
Key wordsITC AFFINImeter Low molecular weight heparin Pentasaccharide Antithrombin Data analysis Heterogeneous mixtures Anticoagulant activity Biosimilars
The development of this protocol has been part of a work carried out with the pharmaceutical company Laboratorios Farmacéuticos Rovi S.A. (Madrid, Spain). We thank them for providing the LMWH, UFH, and AT samples. We also thank Prof. Robert Linhardt and Prof. Fuming Zhang (Rensselaer Polytechnic Institute, Troy, NY) for providing UFH.