Elsevier

Carbohydrate Polymers

Volume 203, 1 January 2019, Pages 87-94
Carbohydrate Polymers

Characterization of epimerization and composition of heparin and dalteparin using a UHPLC-ESI-MS/MS method

https://doi.org/10.1016/j.carbpol.2018.08.108Get rights and content

Highlights

  • A novel UHPLC-HILIC/WAX-MS/MS approach was applied to characterize HONO depolymerized heparin/dalteparin.

  • Ambiguous disaccharide identification was achieved with unique standards.

  • Isotopic labelling was successfully applied for the sameness evaluation of generic dalteparin.

  • Hexuronic acid epimers were identified by MS/MS/MS (CID and HCD) with LTQ-Orbitrap.

Abstract

Heparin is a significant anticoagulant that has been used in clinic over decades. Although numerous efforts have been made to characterize the molecular structure of heparin and its derivatives for safety of the medicine, technical barriers still exist because of their structural complexity. In this study, we have established a method capable to evaluate both the epimerization and composition of heparin and dalteparin by a UHPLC-HILIC/WAX-MS/MS approach. Ten major disaccharide building blocks retaining the epimerization configuration of parental heparin chains were generated and well separated, 9 of which were unambiguously identified. Isomer identifications were achieved through high-resolution tandem mass spectrometry analysis with reference to elaborately prepared standards. The method was successfully applied for the sameness study of generic dalteparins in combination with an isotopic labelling procedure. We believe this robust method maybe adapted to quality control in pharmaceutical production of heparin and LMWHs.

Introduction

Heparin is a polydisperse polysaccharide extracted mainly from pig intestine mucosa. The heparin polysaccharide chains are linear and polyanionic, with repeating disaccharide units of α-L-iduronic acid (IdoA) or β-D-glucuronic acid (GlcA) residue 1→4 linked to glucosamine (GlcN) residue. The sugars are modified by N-, 6-O- and 3-O-sulfation on the GlcN residues as well as 2-O-sulfation on the hexuronic acid. The 3-O-sulfation is critical for heparin to form a specific pentasaccharide domain that specifically bind to antithrombin with high affinity, which is essential for its anticoagulant activity (Lindahl, Backstrom, Thunberg, & Leder, 1980). Recent publications also elucidated the importance of epimerization on the anticoagulant activity (Das et al., 2001; Wang et al., 2017).

The biological activity of heparin products can be efficiently measured by established methods, while determining the molecular structure of heparin has always been challenging due to its extreme heterogeneity. However, appropriate and comprehensive characterization is essential to guarantee its safety as a medicine, because heparin can be easily contaminated by other glycosaminoglycans, leading to life-threaten consequences (Blossom et al., 2008). To prevent occurring such a crisis again, global experts and authorities are making big effort to develop and reinforce multiple dimensional methods for controlling the quality of heparin products (Szajek et al., 2016).

To reach a complete structural characterization of heparin, multiple parameters are examined, including size distribution, overall charge density, composition and sequence of the polysaccharide. Analysis of disaccharide composition is one important part for quality control of heparin.

Disaccharides can be generated by enzymatic or chemical depolymerization of heparin. Combined treatment with heparin lyases (I + II + III) results in almost complete depolymerization of heparin, enabling recovery of total di and tetrasacchrides of the polysaccharides. However, beta-elimination by the enzymes leads to the generation of unsaturated Csingle bondC double bonds between C4 and C5 at the non-reducing end of the hexuronic acid residues (Yang et al., 2011; Yang, Chang, Weyers, Sterner, & Linhardt, 2012), destroying the original epimerization conformation of the C5 carboxyl group. The classical chemical depolymerization by treatment with HONO at low pH depolymerizes heparin, resulting in partial depolymerization while maintaining epimerization information of the parental chains (Conrad, 2001). Resulting disaccharide species are illustrated in Fig. 1A.

For analysis of the disaccharides, chromatographic separation has been the major means, e.g. strong anion exchange (SAX) (Miller et al., 2016), reverse phase ion pairing (RP-IP) (Yang et al., 2011), and hydrophilic interaction chromatography (HILIC) (Sun et al., 2017), that couples with diverse detection methods, including radio-lableing or post-column fluoresce labeling of the disaccharides, and UV-absorption (for enzyme degraded disaccharides).

Electrospray ionization (ESI)-MS is a powerful tool for structure characterization of heparin, especially when coupled with liquid chromatography (LC) (Kailemia, Ruhaak, Lebrilla, & Amster, 2014; Zaia, 2009). It’s superiority in sensitivity and high capability to analyze mixtures makes it an ideal tool for heparin analysis. Tandem mass spectrometry (MS/MS) can be used to identify mono-saccharide composition as well as the location of modification groups. A high resolution mass spectrometer is regularly desired for MS/MS or MS/MS/MS experiments. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, when coupled with collision induced dissociation (CID) (Kailemia et al., 2013), electron detachment dissociation (EDD) (Wolff, Amster, Chi, & Linhardt, 2007), or negative electron transfer dissociation (NETD) (Wolff et al., 2010), provide robust capability for structure elucidation, especially for epimer identification. FT-Orbitrap is an alternative, and more accessible option to perform MS/MS or MS/MS/MS analysis. The CID mode is commonly applied, with helium as collision gas. The high energy collision dissociation (HCD) mode, with nitrogen as collision gas, is more often seen in proteomic studies (Nilsson, 2016).

Low molecular weight heparins (LMWHs) are fragmented heparin sharing similar functions, but with several advantages because of improved bioavailability, predictable anticoagulation activity, ease of administration, and no need for monitoring (Quader, Stump, & Sumpio, 1998). The backbone structure of LMWHs is identical to heparin, with specific structures at their terminus (Lever, Mulloy, & Page, 2012). Several uncommon modifications on LMWHs were also reported(Sun et al., 2016; Wang et al., 2018). Dalteparin (Fig. 1B) is one of the LMWH products manufactured through HONO fragmentation of heparin followed by borohydride reduction, resulting in the formation of a 2,5-anhydro-mannitol ring at the reducing end (Guo & Conrad, 1989). To determine the molecular structure of dalteparin, Zaia et al. (Zaia et al., 2016) and Bisio et al. (Bisio et al., 2015, 2017) applied MS, triple detector array (TDA) detectors, and NMR, respectively, coupled with separation techniques to acquire comprehensive analytical data. However, most of the disaccharide studies (Sun et al., 2017; Wang, Buhse, Al-Hakim, Boyne Ii, & Keire, 2012) used a single or mixture of heparin lyases to digest dalteparin, which can not provide the epimerization information of the disaccharides, loosing significant information of the parental polysaccharides.

In this study, we have combined the robust UHPLC-HILIC/WAX-MS/MS method with HONO digestion of heparin for the characterization of its epimerization and composition. Further, we have established a novel approach by reduction with NaBD4 following HONO digestion, which provides additional information regarding the production process of dalteparin. To verify the method, we analyzed 4 different dalteparin samples, one original (Fragmin) and three generic products. The results proved that our approach is a high resolution quantitative method for the analysis of heparin disaccharides, which has the potential to be adapted for quality control of heparin and heparin derived medicines.

Section snippets

Materials

Three porcine heparin samples were provided by different suppliers. O-desulfated heparin was prepared by solvolytic desulfation with dimethyl sulfoxide as described (Nagasawa, Inoue, & Kamata, 1977). Injectable low molecular weight heparin, Fragmin and 3 generic dalteparin were obtained from pharmacy, China. Fondaparinux Sodium was acquired from Sigma-Aldrich (St.Louis, USA). Recombinant D-glucuronyl C5-epimerase (Hsepi) and heparan sulfate 6-O-sulfotransferase (6-OST) were purchased from

Analysis of heparin disaccharides

Analysis of disaccharide building blocks is one of the major means for characterization of heparin and LMWH structure. So far the majority of efforts has been devoted to analyze the total population of disaccharides derived by enzymatic depolymerization, which has a drawback of losing epimer conformation of the hexuronic acid at the non-reducing end. Our study made an effort to analyze the disaccharides derived by HONO depolymerization of heparin, which preserves the authentic structure of the

Conclusions

In summary, different from previous reported approaches by RP-IP (Babu et al., 2011), porous graphitic carbon (PGC) (Gill et al., 2012) and HILIC (Gill, Aich, Rao, Pohl, & Zaia, 2013), our UHPLC-HILIC/WAX-MS/MS method separated and detected essentially all the disaccharide species in HONO depolymerized heparin/dalteparin. Using the disaccharide samples of HS isolated from mutant mice in combination with exo-glycosidase treatment, we identified the rare component of G2SM that has not been

Acknowledgements

This work was supported by grants fromthe National Key R&D Program of China (2017YFF0205404), the National Sharing Platform for Reference Materials in China, and the Swedish Research Council (2515-02595).

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