Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Abstract

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-O-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production.

Analysis of heparin oligosaccharides by capillary electrophoresis–negative-ion electrospray ionization mass spectrometry

“…This CE-MS method was useful for disaccharide compositional analysis, bottom-up analysis, and top-down analysis. The results obtained were comparable or better than those obtained when these analyses were performed using LC-MS[4, 8, 9]. Moreover, when compared to our previously published, normal polarity CE hyphenated positive-ion MS, the use of reverse polarity CE hyphenated negative-ion MS should facilitate its application for CID tandem MS to determine sequence of chains within LMW heparins or their lyase treated products.…”

“…S4), are observed as disaccharide and tetrasaccharides[10]. In addition to normally encountered building blocks, a linkage region tetrasaccharide was also identified[9]. Moreover, 3- O -sulfo group containing tetrasaccharides that are remnants of the antithrombin pentasaccharide binding site, such as the one present within the structure of Arixtra®, can also be determined by CE-MS.…”

“…Ions with ammonium adduction are also observed and are shown in Figure 6A. CE-MS analysis could be completed in less than 30 min with a lower level of ammonium adduction than observed using hydrophilic interaction liquid chromatography (HILIC)-LC-MS[9], which results in less false positives, simplifying the data processing.…”

“…There are three types of routinely performed heparin and LMW heparin structural analysis that utilize hyphenated MS methodologies, disaccharide compositional analysis [4, 10, 11], bottom-up analysis [9, 12], and top-down analysis [8, 13]. In the case of disaccharide bottom-up analysis and compositional analysis, heparin sample is first treated with one or more heparin lyases [4, 9–12] to afford either oligosaccharides or disaccharides for subsequent LC or CE separation and MS analysis. Top-down analysis examines the whole intact GAG chain and has been limited to either small GAG chains, such as bikunin [19], or LMW heparins[8, 13].…”

GlycCompSoft: Software for Automated Comparison of Low Molecular Weight Heparins Using Top-Down LC/MS Data

“…LMW heparins, including innovator drugs and more recently generic versions, have undergone a great deal of regulatory scrutiny because of their polycomponent nature and their high level of structural complexity [2]. Studies of new approaches to determine the structure of LMW heparins have included bottom-up [3], top-down [4] and combined liquid chromatography (LC)-mass spectrometry (MS) analysis [5–6]. Despite these major advances, the rapid and accurate glycomic analysis of glycosaminoglycans by LC-MS has been plagued by the absence of suitable bioinformatics software.…”