Analysis of sulfates on low molecular weight heparin using mass spectrometry: Structural characterization of enoxaparin

Abstract

Introduction:

Structural characterization of Low Molecular Weight Heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly Enoxaparin.

Areas Covered:

This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as “enoxaparin”, “mass spectrometry”, “low molecular weight heparin”, “structural characterization”, etc.

Expert Commentary:

This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.

Introduction

Heparin is a molecule belonging to the family of Glycosaminoglycans (GAGs), and consists of the heterogeneous mixture of polymers ranging from 5,000–30,000 Da. Due to the presence of heterogeneity, this molecule has been referred to as “Unfractionated Heparin (UFH)” amongst clinicians and researchers [1]. Heparin possesses biological functionality towards angiogenesis and host-pathogen interactions [2,3,4]. Furthermore, Heparin is popular in the pharmaceutical industry for its anti-coagulant properties, and its depolymerized version, termed Low Molecular Weight Heparins (LMWH), has gained much attention in the recent past. Different types of LMWH are derived based on differing depolymerization processes. These molecules are similar to UFH in monosaccharide composition and oligosaccharide sequence. LMWH possess several advantages over UFH due to their lower molecular mass, including prolonged antithrombotic effect and better bioavailability. Given that LMWH do not bind to plasma proteins and endothelial cells, they have a longer half-life in circulation [5].

Due to the often-reported side effects causing Heparin Induced Thrombocytopenia (HIT), LMWHs have been explored as anticoagulants [6]. These molecules are considered more potent compared to unfractionated heparin (UFH) [7]. Herein, the choice of depolymerization process dictates the composition percentage of pentasaccharide units involved in antithrombin activity. It has been shown that the LMWH having longer chains have higher antithrombin activity compared to shorter chains [8]. This indicates a major challenge in associating functional relevance to LMWH from its heterogeneous polysaccharide structures. Understanding charge distribution from the varying sulfation, carboxylation units on specific saccharide provides knowledge on active pharmaceutical ingredient (API) [9,10]. The chemo-enzymatic process resulting in depolymerization of heparin also becomes important to study due to its regulatory effect on physico-chemical properties. Amongst currently marked LMWH, the present review focuses on Enoxaparin as it is used to treat a multitude of disorders. Enoxaparin has the highest Anti-Xa: Anti-IIa ratio and its plasma half-life is second only to Tinzaparin. Enoxaparin also reports the highest concentration of free Tissue Factor Pathway Inhibitor (TFPI) when injected subcutaneously [11].

As Enoxaparin is one of the most commonly prescribed LMWH for many disorders [12,13,14,15,16], it becomes critical to identify methods that could be used to determine the sameness of biosimilarity in enoxaparin compared with that of an innovator molecule. In this regard, FDA guidelines suggest five different criteria for demonstrating sameness of an in-house product to that of an innovator molecule [17]. In addition to biological function (phenotypic sameness) and in vivo pharmacodynamics, the following listed characterization instruments are used to associate sameness. Various analytical techniques include but are not limited to RPIP-LC-MS (Reverse Phase Ion Paired – Liquid Chromatography – Mass Spectrometry), HILIC-MS (Hydrophilic Liquid Chromatography-Mass Spectrometry), SAX (Strong Anion Exchange Chromatography), CZE (Capillary Zone Electrophoresis), and SEC (Size Exclusion Chromatography) have been recently adopted (Fig. 1). Combinatorial usage of instruments has also been applied for mass spectral characterization of LMWH. Thus, this review focuses primarily on structural verification and attempts to identify a strategy that may be used to comprehensively characterize sulfates on LMWH, mainly Enoxaparin (Table I).

Figure 1:

The schema here describes various parameters that need to be evaluated using various instrumentation approaches to validate LMWH. Also, shown herein are different tests performed for method validation. These aspects are covered for evaluating sameness for any biosimilar or generic molecule.

Table I:

Physico-chemical properties of Heparin

Heparin Type	Molecule Name	Molecular Weight (kDa)	Characteristic structural feature
Unfractionated Heparin	Heparin	8 – 15	IdoA-2S-(1→4)-GlcNS,6S
Low Molecular Weight Heparin	Enoxaparin	4.5	4,5 uronic acid on non-reducing end; 1,6-anhydro ring on the reducing terminus (15 – 25 %)
Low Molecular Weight Heparin	Dalteparin	6.0	anhydromannitol ring at the reducing end
Low Molecular Weight Heparin	Tinzaparin	6.5	Non-reducing end with unsaturated uronate residue

Heparin structure and its implications for antithrombin activity

Heparin is a polysaccharide with characteristic trisulfated disaccharide units of IdoA2S-GlcNS6S. Its synthesis begins via chain extension of a tetrasaccharide linker O-linked to serine residue on a protein backbone [18]. In the presence of nucleotide-sugar substrates such as uridinediphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridinediphosphate-glucose (UDP-Glc), monosaccharide addition occurs via the exostosin glycosyltransferase (EXT) enzyme [19]. Heparin enhances ~1000-fold anticoagulation activity of antithrombin-III (AT) through complex formation. This interaction results in the inhibition of two coagulation proteases, thrombin (Factor IIa) and Factor Xa (FXa) [20]. This catalytic action by Heparin-bound AT is facilitated by the presence of a pentasaccharide unit as a part of heparin’s polysaccharide backbone. This unit consists of carbohydrates with varying degrees of sulfation, and is represented as: GlcNAc6S-GlcA-GlcNS3S6S*-IdoA2S-GlcNS6S (abbreviated as ANAGA*ISA). The presence of 2-O-sulfate on IdoA (Iduronic acid) monosaccharide increases affinity towards AT, whereas the presence of 6-O-sulfate on glucosamine indirectly contributes to AT binding by proportionately increasing the ²S₀ conformation [21]. Because LMWH are derived from Heparin, the structure-function analysis of Heparin would help to establish the functional role of heterogeneous chains present on LMWH. Moreover, an in-depth structure-function analysis of Heparin would also help to identify key design strategies for synthesizing Heparin mimetics beyond LMWH. Initial scientific steps have already been taken in this direction [22]. Polymer Chain and Extension Units on LMWH

Enoxaparin is derived from Heparin, extracted from porcine intestinal mucosa, by depolymerization that leads to lower molecular weight fractions. Enoxaparin is further obtained via alkaline β-elimination of the UFH benzyl ester. By contrast, the other two LMWH, i.e. Dalteparin and Tinzaparin, are chemically derived by nitrous acid depolymerization and enzymatically by heparinase from F. heparinum, respectively [23]. Enoxaparin contains variants of uronic acid on its non-reducing end, whereas the reducing end consists of a 1,6 anhydro closed ring-like structure, or possesses an open conformation consisting of a hydroxyl group [24]. The non-reducing end contains either 4,5 unsaturated or a saturated form of uronic acid. These terminal groups are flanked by a repeating unit of GlcNAc (and variants) and IdoA/ GlcA with β1,4 linkage, which extends from n=1 to 22. The degree of sulfation on the repeating unit primarily determines the potency of LMWH and its binding to Factor Xa/IIa in an in vitro assay [25]. For example, a compositional analysis of acetyl groups, sulfate to carboxylate ratio, 1,6 anhydro, active pentasaccharide unit determines sameness between the generic and innovator molecule. Due to its inherent complexity and inbuilt heterogeneity in the structural composition of Enoxaparin, it is indispensable to conduct a comprehensive analysis of its characterization. In this regard, structural characterization between the biosimilar and innovator Enoxaparin will be required to meet regulatory guidelines for generic drug approval.

Latest therapeutic uses of Enoxaparin

Enoxaparin is majorly used to treat Deep Venous Thrombosis (DVT) and pulmonary embolism (PE) which constitutes Venous Thromboembolism (VTE). In regard to treatment, adequate dosages of Enoxaparin have still not been finalized. There are patients for whom 40mg dosages of Enoxaparin are given once or twice daily, or 0.4 – 0.5 mg/kg daily dosages are also recommended. A recent study by Pannucci et al. attempted to compare Enoxaparin dose response in the plastic and reconstructive surgery population [26]. Baumgartner et al. presented yet another study wherein they suggest that prophylactic dosages of Enoxaparin may not serve patients who undergo abdominal cancer surgery. Given that cancer results in VTE due to malignancy and major surgery, anticoagulants find tremendous uses in the treatment of such patients. But the right dosage of Enoxaparin is essential for the treatment of VTE in cancer patients, which this study found to be inadequate [27]. Alnatsheh et al. further studied the occurrence of VTE in obese and non-obese patients who were given prophylactic dosages of Enoxaparin. In this study, no differences were observed in VTE occurrence between the two cohorts, but the authors suggest conducting randomized clinical trials to confirm these findings [28]. Due to the popularity of Enoxaparin in treating VTE, it has become critical to perform randomized clinical trials of various diseased conditions to identify disease-specific dosages for effective administration. Thus, collective efforts by clinicians, researchers and doctors will be required to determine the right dosages under different circumstances, and finally to individualize Enoxaparin use amongst patients.

Heparinase digestion of LMWH

The digestion protocol resulting in Heparin depolymerization to LMWH is as illustrated in Table I. Three different types of heparinase results in specific cleavage onto the repeating unit of LMWH [29]. Heparinase obtained from Flavobacterium Heparinum (F. Heparinum) primarily cleaves between GlcN and uronic acid (Iduronic acid/Glucoronic acid) [30]. Heparinase I and III cleave to LMWH based on the decreasing degree of sulfation, whereas Heparinase II cleaves almost at all sites [31,32,33,34,35,36]. Thus, the choice of Heparinase II for digestion appeals as regards introducing homogeneity in molecular fragments. However, this simultaneously results in a highly fragmented species that is much more tedious to characterize. Heparinase obtained from Prevotella Heparinolytica (P. Heparinolytica) also efficiently cleaves LMWH similar to Heparinase I from F. Heparinum. Furthermore, F. Heparinum contains Heparinase II that interferes with the cleavage of LMWH treated with Heparinase I. Such interference is not observed when Heparinase from P. Heparinolytica is used, due to lack of other enzymes present in the organism [37]. However, the K_M for cleavage of the latter is slightly larger than that obtained for Heparinase I from F. Heparinum. Thus, to obtain complete digestion, it is indispensable to have an enzyme which possesses high V_Max and low K_M properties. Furthermore, in regard to reaction conditions, the enzymatic buffer resulting in the cleavage requires Bovine Serum Albumin (BSA) as the control protein. While buffer conditions can be adjusted such that complete digestion results in disaccharide, tetrasaccharide, and hexasaccharide fragments, the presence of extraneous protein causes contamination. Thus, additional sample preparation steps can be incorporated involving treatment with either acetone or ethanol for protein precipitation. These steps may be added after digestion of heparinase such that the carbohydrate is finally suspended in a protein-free buffer solution for sameness evaluation.

Regulating Sulfation for Structural Characterization

Due to its efficacy and anticoagulant properties, Enoxaparin is popular as an LMWH-based therapeutic drug (Fig. 2). Moreover, Enoxaparin digestion by Heparinase II results in characteristics tetrasaccharide fragments, including, but not limited to, deltaUA-GlcNS6S-GlcA-GlcNS3S, deltaUA-GlcNS6S-GlcA-GlcNS3S6S, deltaUA-GlcNAc6S-GlcA-GlcNS3S6S, and deltaUA-GlcNAc-GlcA-GlcNS3S. The disaccharide structure mostly released contains deltaUA2S-GlcNS6S. These characteristic structures exhibit a prominence of sulfate groups on carbohydrates and introduce complexity in structural verification [38,39,40]. Furthermore, the sulfate groups are critical for an anti-coagulant property, and thus are relevant from a functional perspective [9,41,42]. Because sulfation is primarily involved in introducing structural variability in Enoxaparin, the specificity analysis of sulfolyltransferases in catalyzing sulfolyl groups onto various carbohydrates requires assessment (Table II). Each enzyme adds a mole of sulfate per mole of PAPS (3’-phosphoadenosine 5’-phosphosulfate) in a bi-bi substrate reaction. The addition of a sulfate group results in a bulky molecule and reduces heterogeneity that would otherwise have been introduced from variable sulfation (Fig. 3). Taken together, a strategy whereby enoxaparin is treated with sulfolyltransferase enzymes for maximizing sulfation, and thus reducing heterogeneity on carbohydrate substrates, may simplify structural characterization amongst various enoxaparin grades. While this approach will change the overall sulfation patterns on Enoxaparin and result in changes in the native structure, it will nevertheless reduce variability due to sulfation. Such treatments can be applied only when a comparative analysis is performed. For example, biosimilar and innovator Enoxaparin can be treated under the identical enzymatic conditions and compared for their structural identity. Thus, the methodology can be adopted for structurally comparing Enoxaparin obtained from various sources.

Figure 2:

These structures here illustrate two of the tetrasaccharide moieties obtained for Enoxaparin. Particularly, here 2 variants with or without 4,5 unsaturation on the non-reducing terminus of enoxaparin is exhibited. Such highly sulfated structures introduce heterogeniety in characterization.

Table II:

Substrate specificity of sulfolyltransferase acting on GAGs

S. No.	Substrate(s)	Sulfolyltransferase	Enzyme Specificity
1	GlcNAc	NDST1 and NDST2 N-deacetylase/N-sulfotransferase	Specific
2	GlcNS	6OST-1 6O-sulfotransferase1	Non-specific
3	GlcNS6S	3OST-1 3O-sulfotransferase1	Specific
4	IdoA	2OST-1, 6OST-1 2O-sulfotransferase1, 6 O-sulfotransferase1	Specific/Non-specific

Figure 3:

This schema lists products formed in the presence of specific sulfolyltransferases and specific reactant.

Sample preparation of Enoxaparin for Structural Characterization

Sample preparation for LMWH or Enoxaparin has traditionally depended on the source of its origin. Usually, the active pharmaceutical ingredients (API) in Enoxaparin are pressure dialysed against a 1000 Da molecular weight cutoff membrane, and then lyophilized. These samples are then solubilized in the variety of buffers compatible with the enzymatic digestion protocol. For structural characterization, different enzymes such as Heparinase I, II and III are usually pooled together, and Enoxaparin is incubated with them for 16 – 18 h at 37°C. The optimal concentration of Heparinase mixture for treatment means complete digestion of oligosaccharides into disaccharide units [43]. Appropriate sized molecular weight cutoff membrane is further used to remove intact Heparinases from the digested product, or the sample is heated at 100°C for 5 min to quench the reaction [38]. Since the introduction of Heparinase in the final mixture could increase sample noise, it is desirable to use an appropriate molecular weight cutoff membrane to remove these enzymes from the digested product. This procedure will increase the S/N ratio and further clean the experimental output. Downstream analytical applications, such as MS, demand purity in the compound to remove any unwanted m/z signal on the total ion chromatogram (TIC). Thus, an overall strategy whereby contaminating additives are removed serves as a prerequisite for the sample preparation step.

Protecting Sulfation for Intact MS

Dell et al. have identified β-elimination as a chemical methodology for peeling O-glycans from the cell surface or circulatory glycoproteins. While the approach results in the analysis of cleaved O-glycans, a part of the procedure involves modification of –OH groups on carbohydrate sidechains into –OMethyl, such that their damage or loss is reduced during passage from the high-resolution mass spectrometry instrument [44]. Because methyl groups are not cleaved in spite of high ion source temperature, analysis of intact molecules is possible. Similarly, sulfoesters present on the carbohydrate units of LMWH become labile at high temperature, and are prone to the hydrolysis that results in the loss of a side-group. Various protecting groups have been suggested for use in the preparation of sulfoesters, but none has been classified for protecting sulfate groups while running for spectral analysis. Some of these suggested include phenol, trichloroethyl, neopentyl, isobutyl, and others that form sulfate diester [45,46]. However, in the presence of nucleophiles, these sulfate diesters are prone to attack at either the carbon or the sulfur atom, resulting in dissociated molecules. In this context, therefore, the identification of an effective methyl group as a protecting agent for hydroxyl might well work for sulfoesters. Some other binding agents, such as basic peptides, have also been used to circumvent sulfate loss. This has been shown in various studies wherein analysis of a Heparin-peptide mixture resulted in the identification of an intact molecular mass of complex molecules without loss of sulfate [47,48].

Recent advancements in the field of mass spectrometry have also shown Matrix Assisted Laser Desorption/Ionization (MALDI) or its variant, surface-enhance LDI, as a promising tool for reducing sulfate loss. This reduction stems from usage of correct insource conditions, both related to the instrument parameter and that of the buffer employed [49]. However, application of FT-ICR MS-based mass spectrometry has not prevented sulfate loss, but has nevertheless allowed identification of sites of sulfation by conducting cross-ring cleavages, which it is able to do via the negative electron transfer dissociation (NETD) technique employed using FT-ICR MS [50]. Kailemia et al. recently investigated the use of sodium hydroxide as a spray solution to circumvent sulfate loss during mass spectrometry analysis. In this study, exhaustive deprotonation of acidic groups in precursor ions was performed using 1 or 2 mM sodium hydroxide, and resulted in stabilization of sulfo groups in those oligosaccharides which are either lowly or heavily sulfated. This use results in adequate fragmentation of the glycosidic bonds and cross-ring cleavages, in addition to retaining the site of sulfate loss. Overall, this methodology is presented as demonstrating a comprehensive structural characterization for sulfated oligosaccharides [51].

Mass Spectrometry for LMWH

Enoxaparin is a highly emerging LMWH, annotated as a category B pregnancy drug. Typically, enoxaparin sodium contains eight disaccharide units that are analyzed by various chromatographic techniques [52]. For structural verification of enoxaparin or LMWH, various tandem mass spectrometry techniques have been recently used ,including, but not limited to, high performance liquid chromatography (HPLC) with quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and diode array detector (DAD) MS, capillary electrophoresis (CE) with MS, HILIC Fourier transform (FT) MS, ultra performance (UP) SEC-QTOF-MS, RPIP-UPLC-MS, and others. [39,40,53,54,55,56,57]. In a recent study, oligosaccharides resistant to Heparinase II digestion were also analyzed using LC-MS [58]. Li et al. reported a fingerprinting methodology using RPIP-ESI-MS, in which an ion-trap TOF MS was used tandem with capillary HPLC for enoxaparin oligosaccharide assignment. The authors used 15mM pentylamine (PTA) as an MS compatible ion-pairing agent to segregate different Enoxaparin oligosaccharide, and then matched it with in-house developed software. Using this software, the authors report identification of 150 oligosaccharides belonging to enoxaparin [59]. The same group later reported further characterization of Enoxaparin digested separately with Heparinase I, II, and III. In total, 200 different oligosaccharides ranging between a degree of depolymerization (dp) 2 to dp10 were found, using three different enzymatic treatments of Enoxaparin [38]. Liu et al. reported a top-down and bottom-up approach to compare innovator and biosimilar version of Enoxaparin by carefully selecting mass spectrometry approaches. This article covers results wherein intact Enoxaparin chains, Heparinase II digested oligosaccharides were utilized in hydrophilic interaction chromatography (HILIC) Fourier Transform (FT) MS. Completely digested disaccharides were further used in RPIP-LC-ITMS for bottom-up analysis of Enoxaparin drugs. A comprehensive analysis of four Enoxaparin drugs depicted similar structural features. The major difference observed in the top-down approach between these four drugs was due to the difference in the parent heparin used by different manufacturers [60].

In another study published by the same group, a structural correlation was developed between parent heparins and daughter Enoxaparin using an integrated analytical approach. Parent heparin as used by two different manufacturers in the US and Europe were compared here [61]. The same group later identified sameness in the enoxaparin obtained using heparins from bovine intestinal and bovine lung. In vitro potency and structural characterization assays depicted comparable results in enoxaparin obtained from different heparin sources. Thus, the bottom-up and top-down approach adopted by this group helped to establish sameness in enoxaparin obtained from different heparin sources [62,63]. Based on the strategy adopted for deriving LMWH from heparins, their molecular weight (MW) properties will differ. In this context, an article by Ouyang et al. suggests the use of size exclusion chromatography (SEC) inductively coupled plasma (ICP) MS for determining accurate MW of enoxaparin and other compounds. To do this, the authors used SEC-ICP-MS for accurately determining the ion-pairing cation which forms part of the mobile phase. In this work, a specific mobile phase was used as the source of cation and their accurate MW along with the enoxaparin’s MW was successfully determined [64].

The advancement in the field of spectroscopy has enabled precise identification of species of interest from a sample containing a complex mixture. To annotate different polysaccharide fragments present in Enoxaparin, the choice of the right mass spectrometry ionization source serves as a useful initial step (Fig. 4). Accumulating evidence suggests electrospray ionization (ESI) is a useful analyzer due to its soft ionization tendency [65,66]. To this end, use of negative polarity serves to establish correct mass determination of sulfate groups without using an ion-pairing agent. It has also become a general practice to use arixtra as a calibration molecule for optimization of ion source parameters. This molecule consists of octasulfated penta-saccharide moieties, and thus allows for optimized instrument settings for highly sulfated polysaccharides.

Figure 4:

The LC-MS based fragmentation pattern for a 1,6 anhydro terminated enoxaparin grade. This fragmentation profile illustrates different m/z obtained on cleaving enoxaparin into disaccharides, trisaccharides and tetrasaccharides.

Any mass spectrometry-led experiment can be carried out in tandem with liquid chromatography (LC) or by direct infusion. Experiments are initiated by identification of any mixture by first infusing the sample. ESI-led infusion experiments have been used to quantify LMWH disaccharide mixtures, and collisional induced dissociation (CID) has been performed tandem to LC, enabling identification of isomers. Furthermore, ESI-MS tandem to nano-LC has allowed for experiments with nano-litres sample-volume, thus increasing permutations of experiments for optimizing methods [67]. Various other LC use, including reverse-phase ion pairing (RPIP) and Size Exclusion Chromatography (SEC), has enabled identification of complex LMWH or enoxaparin fragments using ESI-MS [68,69]. This has further indicated the usefulness of column compatibility with different ion pairing agents, buffer conditions, solvent requirement, and the like. Such analysis overall has resulted in qualitative identification of molecules. Furthermore, specifically for Heparin Sulfate (HS), synthesis of a labeled molecule such as ¹³C/¹⁵N has enabled quantitative estimation of this GAG [70].

Various other LC-MS methods have been employed for qualitative and quantitative estimation of LMWH species. In addition to the RPIP and SEC, HILIC provides a chemically modified matrix wherein the species binds to the column based on charges [71]. In this case, the mobile phase contains a water gradient with decreasing organic phase content. HILIC LC-MS has also been employed using a chip-based LC/ MS interface for analysis of CS/DS [72]. Unlike HILIC, graphitized carbon chromatography (GCC) LC-MS produces high resolution from its ability to withstand the wide range of pH and physio-chemical conditions [73]. In this regard, the negative ion mode GCC has been employed for heparinase digested heparin or LMWH. Given that Heparin or LMWH contains diverse sulfate groups, an introduction of negative charge is handled using high pH conditions in the presence of ammonium buffers [74].

Exhaustive structural characterization may for analysis also require use of more than one column connected in tandem with MS. Because LMWH isomer analysis is of considerable interest, using multiple LC columns together with MS has enormous potential. To this end, an independent study subjected a digested HS sample to an SEC column, and thereby onto Strong Anion Exchange (SAX) chromatography for wholesome separation. MS analysis resulted in two different isoforms of a hexasaccharide but with a common set of m/z values [75].

Post-experiment Processing for Spectral Analysis

Identification of LMWH composition benefits exploring their biological and therapeutic potentials. The biological and therapeutic potential of LMWH will benefit by an exploration and identification of its composition. The application of various mass spectrometry instruments, such as ESI and MALDI, has revolutionized structural characterization of complex molecules such as Heparin and LMWH [76]. Furthermore, an addition of tandem LC features has already dramatically improved sample analysis for a given set of carbohydrates. However, a major challenge in using tandem LC-MS for GAGs arises from their ability to lose sulfate because they are collisionally-activated molecules. It has been suggested that the application of various ion modes and/or adducts that result in differing charge and adduct states may reduce sulfate loss. This ability prevents or simplifies complexity arising in the fragmentation pattern, but together makes data interpretation highly cumbersome. Towards this end, deconvolution has been proposed as an algorithm that simplifies data interpretation [77]. Deconvolution results in merging various charges states (z) for a given molecular mass (m). m/z for the same sample further differs from multiplying differing absolute masses (m) due to adduct addition or loss. In the former case, assignment of z from various isotopic peaks is simple in cases where z < 10 because the reciprocal of spacing between adjacent peaks provides z. In the latter case, building a theoretical database is required for knowledge of various m/z or intact masses either with or without adduct. For z > 10 wherein the required mass resolution < 3 ppm at an m/z of 1000, the instrument-specific z interpretation becomes tedious [78]. To relieve this problem, various deconvolution or pattern recognition techniques are employed. In an independent study, Fourier transform, Patterson function, and a combination of these were used to automate determination of various charge states (z). This required information obtained from the isotopic peak envelopes to calculate the spacing between peaks with high accuracy [79]. For using deconvolution methods, Decon2LS provides an open-source software package for automated progression of mass spectral data, whereby deisotoping masses using the THRASH (Thorough High-Resolution Analysis of Spectral by Horn) algorithm can be performed.

Software tools for LMWH Spectral Interpretation

With increasing interest in the field of mass spectrometry, many software packages have been developed over the last two decades. The glycoworkbench software can draw structures that correspond to glycans, GAG, etc. It draws mass composition of structures modified with methylation, and implies structures that are permethylated and modified on the hydroxyl-end. glycoworkbench also allows annotation of carbohydrate composition (MS) and product ions (MS/MS) from tandem mass spectra [80]. Glycomod program does carbohydrate annotation using mass spectral data but is not automated [81]. The Heparin oligosaccharide sequencing tool (HOST) is another software package that provides automated data interpretation, but is limited in its ability to analyze homogeneous samples and highly digested LMWH [65]. GlycReSoft open-source software is specific to glycan analysis, but ranks peaks based on the composition of multiple charged MS ions. It is limited in that it cannot analyze oligosaccharide sequence, in that MS/MS product ion spectra are not accounted for [82]. Recently, Wang et al. designed another software, GlycCompSoft, which takes input data from GlycReSoft and computes results within minutes. It is advantageous over GlycReSoft because the labour-intensive annotation of output is completely automated in this software [83].

In comparison, software such as MyriMatch and GAG-ID have been developed for automated data interpretation of complex molecules. Various scoring algorithms have been proposed for automating spectral data, including cross-correlation, hypergeometric distribution, Poisson distributions, Bayesian statistics, dot products, and many others. Most of these algorithms function by matching product ions from either peptide or glycans to that of the observed or database-contained spectrum without distinguishing major versus minor peaks. Software packages such as GAG-ID and Myrimatch use a novel algorithm of multivariate hypergeometric distribution [84]. This analysis relies on distributing peaks found in the spectra in intensity classes such that the known or reported m/z can be categorized based on intensity fingerprints. For GAG-ID, the advantage of multivariate hypergeometric distribution is readily apparent in the way the authors designed a theoretical sequence database to contain every possible derivation of Heparin and HS. GAG-ID further provides a user-friendly GUI and makes feasible high-throughput sequencing of LMWH [85]. Together, amongst known software packages currently available, GAG-ID identifies enoxaparin structures with greater ease.

Conclusion

Amongst known LMWH, Enoxaparin is the most promising anticoagulant, owing to its prescribed utility over Dalteparin and Tinzaparin. Although Enoxaparin is preferred over the other two LMWH, their functional efficacy is not entirely different. In their efficacy towards Factor Xa/IIa, the degree of sulfate-to-carboxylate ratio acts as an important parameter. Thus, in this review sulfolyltransferases and heparinase are recommended to reduce heterogeneity on Enoxaparin and digesting substrates for structural characterization. Following USFDA guidelines, the application of various analytical instruments for sameness evaluation is simplified because the sample becomes homogeneous and enzymatic treatment introduces ease of analysis.

Amongst known instruments of choice, mass spectrometry is popular due to its ability to perform structural characterization and compositional breakup of LMWH. Such mass spectrum evaluation can be obtained by using either ESI or MALDI instrument with various chromatography techniques. Various LC, including SEC, RP, RPIP, HILIC, SAX, CE, and GCC, have been proposed for use. Because compositional analysis of sulfates is also required for understanding key API amongst the pool of heterogeneous mixtures, automation of spectral data introduces yet another ease of functioning. Here, various online tools are referred to, with GAG-ID most prominent for LMWH analysis. Mass spectrometry instrumentation and data interpretation techniques are evaluated in this review as a comprehensive way to structurally analyze Enoxaparin.

Expert Commentary

Low Molecular Weight Heparins (LMWH) are structurally complex, heterogeneous, polydisperse, and highly charged mixtures of chemically or enzymatically treated Unfractionated Heparin (UFH). Due to their less adverse side effects, LMWHs have seen precedence in many thrombotic disorders over UFH. Owing to these factors, LMWHs have been evaluated in multiple randomized clinical trials and found to be safe and effective for the treatment of venous thrombosis, pulmonary embolism, and unstable angina. For patient treatment, they have been found to be equally effective as UFHs, the current treatment choice globally for venous thromboembolism. However, specifically for LMWH, the major caveat in clinical management is that LMWH cannot be tried on patients with a history of heparin-induced thrombocytopenia (HIT), osteopenia, and or on those with LMWH allergy [86]. Moreover, since LMWH are less potent than UFH or Heparin, there is no standardization or dosage information available for their clinical use. The longer-term treatment of LMWH has also not been established, and thus its acceptance as a replacement for UFH or Heparin is limited.

These are the current clinically relevant limitations in promoting LMWH as a therapeutic. Ongoing efforts in this direction will certainly ameliorate these factors, and currently are an area of active research. The overall role of LMWH is very promising in thrombotic disorders, and thus it is of keen interest to keep working in this area for the coming years. Many new pharmacology firms have begun to develop drugs biosimilar to LMWH, and some have succeeded in marketing them in developing nations. Certainly, since biosimilar demands extensive physico-chemical, structural, functional, pharmacokinetic, and pharmacodynamic characterization, it is of the utmost importance that researchers continue to develop tools that are user-friendly and provide extensive characterization of LMWH. Owing to the inherent complexity of its structural features, the use of advanced analytical techniques such as mass-spectrometry (MS) becomes indispensable. In this regard, this review highlights the use of MS in analyzing LMWH. With the advent of high resolution and highly sensitive MS, there is a better understanding of the structural features of LMWH. The FDA previously approved a method for assessing the structural features of LMWH using low-resolution MS, but currently available instrumentation has enhanced our understanding of the structural characterization of LMWH [43]. Major progress has been achieved on account of the optimization of the enzymatic digestion protocol and the development of liquid chromatography (LC) methods. Since MS is mostly used with LC, optimal LC methods are critical for gathering structural attributes. Here, major advancements in MS research will drive a better understanding of the composition of these LMWH and help assess their biosimilarity to innovator drugs. The greatest emphasis should be laid on advanced analytical techniques for comprehensive characterization of these biosimilars, such that more and more pharma companies are able to commercialize these products. This will not only make the drug cheaper (the cost of LMWH is high), but make them more accessible to developing nations as well. Thus, automation of MS data and user-friendly interpretation will provide an integrated approach to MS data analysis and will drive characterization of biosimilar LMWH for drug develpment.

Five-year Viewpoint

Low molecular weight heparins have long been prescribed as an alternative to UFH due to the multiple advantages it carries. Amongst known LMWH, Enoxaparin is considered to be the most-prescribed LMWH as an anticoagulant, both in the US and Europe. Despite having major advantages over the UFH, very few LMWH biosimiliars are available in the market. In the near future, a few if not many biosimilars for LMWH, including Enoxaparin, Dalteparin, and Tinzaparin, will be commercialized, thus further reducing cost per dosage. This will require Himalayan efforts from the pharma industry to demonstrate sameness in the analytical assays and potency tests of these biosimilars. Because currently there are not major available biosimilars for LMWH, there likely are not many well-developed methodologies available globally that can be used to determine sameness of a biosimilar to an innovator drug. In our opinion, more user-friendly techniques employing simpler instrumentation will be developed in the near future. There are various HPLC and tandem MS techniques currently used for the structural analysis of Enoxaparin and other LMWHs. With the advent of ESI-MS, major challenges in the comprehensive structural characterization of sulfates and carboxylates on LMWH are being circumvented. More literature will be available illustrating the identification of sulfates and carboxylates on LMWH using MS. Major emphasis is currently given to Enoxaparin due to large market share, and therefore other LMWH will also be comprehensively characterized for their unique sulfation and carboxylate patterns. Current MS techniques also use either high-end MS instruments such as Orbitrap, or use ion-pairing agents that contaminate MS instrument. Thus, better methods will be developed without affecting instrument performance, simultaneously providing cleaner and more comprehensive structural data for evaluation of sameness. In order to develop an analytical method for complete characterization of chemical structure, better separation techniques of LMWH oligosaccharides are also required. Thus, significant work will be pursued in this direction to completely characterize structures [87]. Given that current efforts are also being directed toward characterization of the monosaccharides, disaccharides, and oligosaccharides involved in LMWH, not much emphasis has been given to the analysis of contaminants using MS. Thus, studies will be conducted using MS and other analytical techniques to determine percentage of contaminants and to structurally determine their composition, so as to identify strategies to rectify them.

Focus will be placed on the changes in clinical practices in identifying the dose of LMWH for individual patients. Current practices include measuring anti-factor Xa and activated partial thromboplastin time (APTT). For LMWH dosing, this method needs to be replaced with anti-thrombin activity to assess the potency of these drugs. Patient-specific dosage and type of LMWH will be further prescribed in the future. Because the thrombin generation capacity of individual patients varies significantly in within a given population, measurement of anti-thrombin activity will be used as a criteria for individualizing the drugs [88].

Another main emphasis will be on the consideration of the effects of alternative medication as anticoagulants in place of LMWH. Because these LMWH drugs are injected, many patients with cancer related thrombosis do not use of them as anticoagulants. The competition from other drugs, such as direct oral anticoagulants (DOACs), will have a significant effect on treatment. Trials have already been conducted using random introduction of LMWH and DOACs in patients to observe their efficacy in treating venous thromboembolism (VTE) and major bleeding [89]. It is easy to see that introduction of such alternative drugs, although important from the patient perspective, will affect overall market share of LMWH as the current standard of care in treatment of VTE. Although this will not affect the role of LMWH in other antithrombotic disorders, relevance in cancer-associated VTE will likely be reduced.

Table III:

Different buffer conditions corresponding to LC-MS instrumentation

LC-MS Type	Condition Used	GAG Studied	Fraction Analyzed
Reversed Phase (RP)	1-phenyl-3-methyl pyrazolone (PMP) derivatization	HS and DS	NR
Reverse phase ion pairing (RPIP)	Dibutylamine as IPA	Unsulfated Heparosan	Upto dp40
Reverse phase ion pairing (RPIP)	Tributylamine as IPA	Unsulfated Heparosan	dp 2 – 20
Reverse phase ion pairing (RPIP)	Tripropylamine as IPA	Partially depolymerized Heparin	Upto 200 components
Hydrophilic interaction (HILIC)	Negative ESI with Ammonium formate modifier	Heparin	Upto dp18
Size Exclusion (SEC)	Use online ion suppressor to reduce ammonium salts	Partially depolymerized CS	Upto dp14
Graphitized Carbon (GCC)	Negative mode with ammonium bicarbonate modifier	Hyaluronan, KS, Heparin and HS	Isomeric disaccharides
Multidimensional LC	SEC and SAX chromatogrpahy	Heparinase treated HS	Hexasaccharide isomer separated
Capillary Electrophoresis (CE)	Reverse CE with Negative ESI	HS	Disaccharide separation

Key issues.

• Enoxaparin is amongst the most well-studied and characterized low molecular weight heparin drugs. There are many studies on its structural characterization.

• The application of enzymes is towards not only enzymatically deriving LMWH (e.g. Enoxaparin) from UFH or Heparin, but also towards having available a sulfated molecular species to reduce structural heterogeneity in the pool of variable compositions of LMWH. Sample preparation techniques are also presented that conserve sulfates on enoxaparin. Such methods are immensely helpful in retaining the structural integrity of these LMWHs.

• Determination of structural composition is presented in the review by advanced analytical technique such as mass spectrometry (MS). Various examples of the use of liquid chromatography (LC) with MS are presented, along with the latest automated software for MS data interpretation.