Application Of Novel Hyphenated Mass Spectrometry Technologies On Low Molecular Weight Heparin Drug Quality Control

Heparin is a structurally complex mixture of sulfated,linear polysaccharide consisting of a disasaccharide repeating unit comprised of uronic acid and glucosamine residues.Heparin exerts its anticoagulant activites by binding to the serine protease inhibitor antithrombing III(AT III)through a specific pentasaccharide sequence.The clinic usage of heparin started at 1930s,its activities in treating venous thrombosis,pulmonary embolism,deep vein thrombosis were soon discovered.Heparin is mainly extracted from porcine intestinal mucosa currently,while after the "heparin crisis" in 2008,United State Pharmacopeial Convention(USP)hosted the 6th Workshop on the Characterization of Heparin Products in 2015 to diverstify the sources of heparin drugs.The reintroduction of bovine heparin into the US market is one of the major discussion during the workshop.Since the bovine spongiform encephalopathy(BSE)disease has been well controlled during the past 35 years,the US Food and Drug Administration(FDA)is also considering bring bovine heparin back into account by setting up a bovine heparin working group,which encourages the study about bovine heparin recently.Low molecular weight heparins(LMWHs)are anticoagulant drugs prepared from unfractionated heparin(UFH)through different kinds of depolymerization procedures.LMWHs have longer half lifes,predictable anticoangulant properties and better bioefficiencies compared with UFH.According to different depolymerization methods,LMWHs with different structures and properties could be generated.Enoxaparin is a kind of LMWHs generated from chemical ?-elimination.Dalteparin and Nadroparin are LMWHs prepared through chemical deamination.Tinzaparin is prepared by enzymatic P-elimination,and parnaparin is prepared by oxidative cleavage.Due to the better properties LMWHs maintain,60%of the UFH is applied to produce LMWH drugs.Enoxaparin is the most widely used LMWH drug occupying 70%of the US LMWH market.The anticoangulant activities of heparin and LMWHs were standardized by World Health Organisation(WHO)by proposing an international standard(IS)with it activity in intemation unit(IU).The pharmacopoeial standards were established according to the IS in different countries,meanwhile guidelines on activity assay and structure characterization were also provided.FDA approved the first generic version of enoxaparin in 2010 and proposed five criterions on the similarity evaluation between generic drugs and innovator drugs,which is equivalence of physicochemical properties,equivalence of heparin source material and mode of depolymerization,equivalence of disaccharide building blocks,fragment mapping and sequence of oligosaccharide species,equivalence in biological and biochemical assays and equivalence of in vivo pharmacodynamic profile.Center for drug evaluation(CFDA)elucidated several aspects on LMWH structure characterization including mososaccharide composition,disaccharide profile,oligosaccharide distribution,oligosaccharide sequence and terminal structures.UFH drugs coming from different biological matirials have different physicochemical properties resulting in different activities and structures.LMWHs are generated from UFHs,thus the relationship investigation between parent heparin and their daughter LMWHs is meaningful in LMWH quality evaluation.Mass spectrometry(MS)is a powerful analytical tool can be coupled with several separating methodologies and provide high sensitivity,high resolution with specificity.High performance liquid chromatography-mass spectrometry(HPLC-MS)and capillary electrophoresis-mass spectrometry(CE-MS)methods were well developed on LMWH structure characterization.Nuclear magnetic resonance(NMR)is another popular technique applied to LMWH quality evaluation.NMR has relatively low sensitivity,however high resolution with the capacity on resolving glucosidic bond,uronic acid and the substitution on glucosamine residues.NMR can distinguish contaminations clearly in heparin or LMWH drugs as well.LMWH characterization strategies can be summarized into two pathways based on the FDA and CFDA requirements,which are the top-down and bottom-up pathways.Be similar with the proteomics strategies,LMWH top-down analysis is basically analyzing samples without any pretreatment.Such as the molecular weight messurement using size exclusion chromatography(SEC),the intact chain fingerprint mapping using LC-MS,the monosaccharide compositional analysis using NMR and the oligosaccharide sequencing by tandem mass spectrometry.The bottom-up strategy requires depolymerization of the LMWH chains.Such as applying HPLC,CE and LC-MS on LMWH disaccharide compositional analysis and oligosaccharide compositional analysis after heparinase treatment.This thesis set up an integrated analytical approach by combining several currently widely used top-down and bottom-up methods on LMWH characterization.The top-down approach includes two aspects,which are the intact chain mapping by hydrophilic interaction liquid chromatography-mass spectrometry(HILIC-MS)and monosaccharide compositional analysis using NMR.The bottom-up approach includes two aspects as well,which are the disaccharide compositional analysis by reverse phase ion-pairing liquid chromatography-mass spectrometry(RPIP-LC-MS)and the oligosaccharide mapping by HILIC-MS.The application of the integrated approach was in 3 levels:(i)Investigate the similarity tolerance of the commercial products by comparing 4 kinds 12 lots commercial LMWH products;(ii)Investigate the difference between LMWHs prepared from bovine intestinal mucosa heparin and bovine lung heparin and discuss the relationship between the bovine parent heparin and bovine daughter LMWHs;(iii)Evaluate 6 pairs of parent heparin and their daughter heparin from 2 different manufactures and obtain the parent-daughter relationship by principal component analysis(PCA).In addition,this thesis also proposed a new negative mode CE-MS method for LMWH characterization,both top-down and bottom-up approach were accompolished on this new CE-MS platform.In this thesis,the main research achievemets are as follows1.An integrated analytical approach based on top-down and bottom-up strategies was established.A strategy for the comprehensive analysis of LMWHs is described that relies on using an integrated top-down and bottom-up approach.LC-MS,an essential component of this approach,is rapid,robust,and amenable to automated processing and interpretation.NMR provides complementary top-down information on the chirality of the uronic acid residues comprising a LMWH.Using our integrated approach four different enoxaparin drugs were comprehensively analyzed.Lovenox(?)and Clexane(?),the innovator versions of enoxaparin marketed in the US and Europe,respectively,and two generic enoxaparins,from Sandoz and Teva,were analyzed.The results,while showing remarkable similarities between different versions of the product and good lot-to-lot consistency of each product,also detects subtle differences that may result from differences in their manufacturing processes or differences in the source(or parent)porcine heparin from which each product is prepared.2.Two kinds of bovine LMWHs were compared using the integrated approach.The worldwide dependence on a single animal species has made the supply chain for heparin and LMWHs quite fragile leading to the search for other sources of these drugs,including bovine tissues such as bovine intestine or lung.The current study is designed to compare LMWH prepared from bovine heparins through chemical ?-elimination,a process currently used to prepare the LMWH,enoxaparin,from porcine heparin.Using top-down,bottom-up,compositional analysis and bioassays,LMWHs,derived from bovine lung and intestine,are shown to closely resemble enoxaparin.3.The relationship between parent heparin and their daughter LMWH was obtained by the integrated approachPrincipal component analysis was applied to normalized abundance calculated in the bottom-up analysis to show parent and daughter correlation in fragment composition.Using these approaches,six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed.Lovenox(?),the innovator versions of enoxaparin marketed in the US was analyzed as a reference for the daughter LMW heparins.The results,showing similarities between LMW heparins from two different manufactures with Lovenox(?)and excellent Iot-to-lot consistency of products from each manufacture,also detects a correlation between each pair of parent and daughter.4.A novel negative mode CE-MS method on LMWH characterization was developedWe examine the analysis of heparin disaccharides,oligosaccharides,an intact low molecular weight using a novel electrokinetic pump-based CE-MS interface.Reverse polarity CE separation and electrospray were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid.The on-line CE hyphenated negative-ion electrospray MS on an LTQ Orbitrap was useful in disaccharide compositional analysis,bottom-up and top-down analysis of low molecular weight heparin was demonstrated.The application of this CE-MS method to the ultra low molecular heparin suggest that the charge state and low sulfate group loss make this method useful in on-line tandem MS analysis of heparins.