•Methodologies for characterizing GAG-protein complexes are reviewed.•Affinity measurement methods and spectroscopic approaches are described.•Advantages and limitations of each method are discussed.Glycosaminoglycans (GAGs) are a family of linear and anionic polysaccharides that play essential roles in many biological and physiological processes. Interactions between GAGs and proteins regulate function in many proteins and are related to many human diseases and disorders. The structural motifs and mechanisms for interactions between GAGs and proteins are not fully understood. Specific bindings, including minor but unique sequences sporadically distributed along the GAG chains or variably sulfated domains interspersed by undersulfated regions, may be specifically recognized by defined domains of a variety of proteins. Understanding the molecular basis of these interactions will provide a template for developing novel glycotherapeutic agents. The present article reviews recent methodologies and progress on the characterization of structural motifs in both GAGs and proteins involved in GAG-protein interactions. The analytical approaches are categorized into three groups: affinity-based methods; molecular docking, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography; and mass spectrometry (MS) techniques. The advantages and limitations of each category of methods are discussed and are based on examples of using these techniques to investigate binding between GAGs and proteins.Download high-res image (180KB)Download full-size image

•Four types of commercial enoxaparins were analyzed by integrated LC–MS and NMR analysis.•Heparinase treatment was used before bottom-up analysis and disaccharide analysis.•Intact chain, oligosaccharide, and disaccharide analyses relied on LC–MS.•Monosaccharide compositional analysis relied on top-down NMR analysis.•Generic enoxaparins and innovator product are similar, differences are observed due to parent heparin and process conditions.A strategy for the comprehensive analysis of low molecular weight (LMW) heparins is described that relies on using an integrated top-down and bottom-up approach. Liquid chromatography-mass spectrometry, an essential component of this approach, is rapid, robust, and amenable to automated processing and interpretation. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues comprising a low molecular weight heparin. Using our integrated approach four different low molecular weight heparins prepared from porcine heparin through chemical β-eliminative cleavage 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 which were supported by analysis of variation (ANOVA), 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.

•The new LC-MRM-MS/MS method can detect the most building blocks of LMWHs.•A maltose modified HILIC column offered the best separation.•The nitrous acid depolymerized LMWHs possess characteristic basic building blocks.•The chemically modified minor components should be carefully examined.Low molecular weight heparins (LMWHs) are important anticoagulant drugs that are prepared through depolymerization of unfractionated heparin. Based on the types of processing reactions and the structures of the products, LMWHs can be divided into different classifications. Enoxaparin is prepared by benzyl esterification and alkaline depolymerization, while dalteparin and nadroparin are prepared through nitrous acid depolymerization followed by borohydride reduction. Compositional analysis of their basic building blocks is an effective way to provide structural information on heparin and LMWHs. However, most current compositional analysis methods have been limited to heparin and enoxaparin. A sensitive and comprehensive approach is needed for detailed investigation of the structure of LMWHs prepared through nitrous acid depolymerization, especially their characteristic saturated non-reducing end (NRE) and 2,5-anhydro-d-mannitol reducing end (RE). A maltose modified hydrophilic interaction column offers improved separation of complicated mixtures of acidic disaccharides and oligosaccharides. A total of 36 basic building blocks were unambiguously identified by high-resolution tandem mass spectrometry (MS). Multiple reaction monitoring (MRM) MS/MS quantification was developed and validated in the analysis of dalteparin and nadroparin samples. Each group of building blocks revealed different aspects of the properties of LMWHs, such as functional motifs required for anticoagulant activity, the structure of heparin starting materials, cleavage sites in the depolymerization reaction, and undesired structural modifications resulting from side reactions.

Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.

•A fingerprinting approach was established for evaluating the sequence of LMWHs.•New software was developed to interpret the complicated MS data of LMWH fragments.•This method is capable to detect special and characteristic LMWH structures.•A total of 94 enoxaparin fragments and 109 nadroparin fragments were identified.•Quantitative comparison between innovator and generic nadroparin were made.Low molecular weight heparins (LMWHs) are linear and highly charged carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. Compared to unfractionated heparin (UFH), LMWHs are prevalently used as clinical anticoagulant drugs due to their lower side effects and better bioavailability. The work presented herein provides a rapid and powerful fragment mapping method for structural characterization of LMWHs. The chain fragments of two types of LMWHs, enoxaparin and nadroparin, were generated by controlled enzymatic digestion with each of heparinase I (Hep I, Enzyme Commission (EC) # 4.2.2.7), heparinase II (Hep II, no EC # assigned) and heparinase III (Hep III, EC # 4.2.2.8). Reversed phase ion pair high performance liquid chromatography (RPIP-HPLC) coupled with electrospray ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) was used to profile the oligosaccharide chains ranging from disaccharides to decasaccharides. A database containing all theoretical structural compositions was established to assist the mass spectra interpretation. The six digests derived by three enzymes from two types of LMWHs exhibited distinguishable fingerprinting patterns. And a total of 94 enoxaparin fragments and 109 nadroparin fragments were detected and identified. Besides the common LMWH oligosaccharides, many components containing characteristic LMWH structures such as saturated l-idopyranosuronic acid, 2,5-anhydro-d-mannitol, 1,6-anhydro-d-aminopyranose, as well as odd number oligosaccharides were also revealed. Quantitative comparison of major components derived from innovator and generic nadroparin products was presented. This approach to profile LMWHs' fragments offers a highly reproducible, high resolution and information-rich tool for evaluating the quality of this category of anticoagulant drugs or comparing structural similarities among samples from various sources.

•A robust RPIP-ESI-MS method was established for LMWHs chain mapping analysis.•Over 200 structures were identified from enoxaparin and nadroparin.•Chain mapping analysis of nadroparin calcium was not reported previously.•Minor impurities were detected using the approach demonstrated herein.Low molecular weight heparins (LMWHs) are structurally complex, highly sulfated and negatively charged, linear carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. They are widely used as anticoagulant drugs possessing better bioavailability, longer half-life, and lower side effects than heparin. Comprehensive structure characterization of LMWHs is important for drug quality assurance, generic drug application, and new drug research and development. However, fully characterization of all oligosaccharide chains in LMWHs is not feasible for current available analytical technologies due to their structure complexity and heterogeneity. Fingerprinting profiling is an efficient way for LMWHs’ characterization and comparison. In this work, we present a simple, sensitive, and powerful analytical approach for structural characterization of LMWHs. Two different LMWHs, enoxaparin and nadroparin, were analyzed using reversed phase ion pair electrospray ionization mass spectrometry (RPIP-ESI-MS). More than 200 components were identified, including major structures, minor structures, and process related impurities. This approach is robust for high resolution and complementary fingerprinting analysis of LMWHs.

Cathelicidins were initially characterized as a family of antimicrobial peptides. Now it is clear that they fulfill several immune functions in addition to their antimicrobial activity. In the current work, three cDNA sequences encoding pheasant cathelicidins were cloned from a constructed bone marrow cDNA library of Phasianus colchicus, using a nested-PCR-based cloning strategy. The three deduced mature antimicrobial peptides, Pc-CATH1, 2 and 3 are composed of 26, 32, and 29 amino acid residues, respectively. Unlike the mammalian cathelicidins that are highly divergent even within the same genus, Pc-CATHs are remarkably conserved with chicken fowlicidins with only a few of residues mutated according to the phylogenetic analysis result. Synthetic Pc-CATH1 exerted strong antimicrobial activity against most of bacteria and fungi tested, including the clinically isolated (IS) drug-resistant strains. Most MIC values against Gram-positive bacteria were in the range of 0.09–2.95 μM in the presence of 100 mM NaCl. Pc-CATH1 displayed a negligible hemolytic activity against human erythrocytes, lysing 3.6% of erythrocytes at 3.15 μM (10 μg/ml), significantly higher than the corresponding MIC. Pc-CATH1 was stable in the human serum for up to 72 h, revealing its extraordinary serum stability. These specific features of Pc-CATH1 may make its applications much wider given the potency and breadth of the peptide's bacteriocidal capacity and its resistance towards serum and high-salt environments.

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin–derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin–derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

•Low molecular weight heparins prepared from different heparin parents were analyzed.•An integrated analytical approach relied on LC-MS and NMR analysis.•Monosaccharide compositional analysis relied on top-down NMR analysis.•Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS.•Differences due to parent heparin were observed using principal component analysis.Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin.