Development Of Analytical Methods For Heparin MW,Composition And Structural Characterization Of Two Kinds Of Heparins

Heparin drugs are the most widely used anticoagulant in clinic,mostly in the field of deep vein thrombosis,hemodialysis,surgery and so on.According to incomplete statistics,global sales of heparin drugs were up to $15 billion in 2018.Heparin is one of the oldest biochemical drugs extracted and purified from animal tissues,especially porcine intestinal mucosa.Heparin is one family member of glycosaminoglycans,and one of the most complex clinical drugs due to its large molecular weight,polydispersity and sequence heterogeneity.However,a lot of analytical issues have been plaguing pharmaceutical analysts between wide applications and structural complexity of heparin.In a word,incomplete understanding of heparin structure limits studies on quality control,new indication expansion and heparin-based new drugs development of heparin drugs.The occurrence of heparin contamination event in 2007-2008 exposed the lack of effective quality control methods for heparin at that time.Since then,development of analytical method for heparin drugs and structural characteristics understanding of heparin is one of the pharmaceutical analysis hotspots.Among them,low molecular weight heparins(LMWHs)widely used in clinic and non-anticoagulant heparins(NACHs)with extensive potential applications are the two most typical heparin products,and their structural studies have become the focus.In our study,systematic researches were carried out on the development of analytical methods for heparin MW,Content and Composition,and structural characterization of heparin,LMWHs and NACHs.Briefly as follows.1.The development and optimization of structural analysis methods for heparin.First,analytical method for heparin MW and content was developed.Size exclusion chromatography(SEC),relying on multiple angle laser scattering(MALS)/refractive index(RI)detectors has been developed for accurate analysis of heparin MW,molecular weight distribution and content in the absence of standards.Compared with the common GPC method,it is no longer limited by the molecular weight standards of the same series of substances.Meanwhile,SEC with inductively coupled plasma/mass spectrometry detectors(ICP-MS)was also applied to quantitative analysis of ion-paired cations from heparin products.This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides.Next,Hydrophilic interaction chromatography with mass spectrometry(HILIC-MS)for heparin disaccharide analysis was developed.HILIC shows good resolution and excellent MS compatibility.Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and semi-quantitatively analyzed.These included common di saccharides,disaccharides derived from chain termini,3-O-sulfo group-containing tetrasaccharides related to anticoagulant activity,along with linkage region tetrasaccharides and their derivatives.Thus,compositional analysis using HILIC-MS can be applied to the field of structural analysis,quality control and process of heparin products.In addition,a novel on-line reverse polarity negative-ion mode CIEF-MS method has been first developed for the separation and analysis based on isoelectric points(pI)of heparin disaccharides even oligosaccharides.CIEF-MS is potentially a powerful tool with high sensitivity for distinguishing the charge heterogeneity of complex mixtures.2.Deep insight into the structure of heparins and LMWHs(the most widely used LMWH in clinic)from different animal sources,manufacturers and production times.The molecular weight distribution,composition and structure of heparin and LMWHs from different animal sources,manufacturers and production times were systematically analyzed and compared.The above results provide deep insight into the structural characterizations of heparin/LMWHs and understanding of the relationship between structural differences and raw animal sources,manufacturers,production times and anticoagulant activity.3.Deep insight into the structure of non-anticoagulant heparin(NACH)from anticoagulant heparin.Comprehensive structural analysis including disaccharide analysis,top-down and bottom-up analysis was applied to obtain deep insight into the structural characterizations of NACH.Meanwhile,the binding affinities of NACH with important heparin-binding proteins are different,which shows wide potential clinical applications of NACH.In our study,the development and application of structural analysis methods for heparin improves quality control methods for heparin products and deep insight into structural characteristics of heparin,LMWHs and NACH were obtained,which is solid theoretical foundation of their production,process optimization,quality control,new indication development and new drugs development.