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Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heparin （Hp） is the most widely used anticoagulant drug in the clinics， with an annual global output of over 10 billion dollars. Hp， a member of the glycosaminoglycans （GAGs）， is prepared from porcine intestinal mucosa via extraction， separation， and purification. Hp is a linear polysaccharide with repeating disaccharide units. Low-molecular-weight heparins （LMWHs） are depolymerized from Hp via chemical or enzymatic degradation. Compared with Hp， LMWHs exhibit less bleeding side effect， milder immunogenicity， and higher bioavailability when injected subcutaneously. In general， Hps， including LMWHs， are high complex drugs with large molecular weights （MWs）， inhomogeneous MW distributions， and structural heterogeneity， including different degrees and locations of sulfonation， and unique residues generated from different production processes. Thus， developing efficient analytical methods to elucidate the structures of Hps and characterize or quantitate their properties is extremely challenging. Unfortunately， this problem limits their quality control， production optimization， clinical safety monitoring， and new applications. Research has constantly sought to elucidate the complicated structures of Hp drugs. Among the structural analysis and quality control methods of Hp currently available， chromatographic methods are the most widely studied and used. However， no literature thoroughly summarizes the specific applications of chromatographic methods in the structural analysis， manufacturing process， and quality control of Hp drugs. This paper systematically organizes and describes recent research progresses of the chromatographic methods used to analyze Hp drugs， including the identification and composition of monosaccharides， disaccharides， oligosaccharides， and polysaccharides. The applications， innovations， and limitations of these chromatographic methods are also summarized in this review. The insights obtained in this study will help production and quality control personnel， as well as drug researchers， obtain a deeper understanding of the complex structures of Hp drugs. This paper also provides a comprehensive reference for the structural analysis and quality control of Hps， proposes ideas for the development of new quality control methods， and lays a strong foundation for the in-depth structural elucidation of Hp drugs.

Heparin, an old but first-line anticoagulant, has been used over a century. It is a heterogeneous, linear, highly sulfated, anionic glycosaminoglycan with a broad distribution in relative molecular weight and charge density. These structural properties allow heparin to selectively interact with multiple proteins, leading to heparin's various pharmacological functions, such as anticoagulant, anti-viral, anti-tumor and anti-inflammatory activities. Clinical data suggest that unfractionated heparin or low molecule weight heparin could decrease mortality in COVID-19 patients with sepsis-induced hypercoagulation through the anticoagulant, anti-viral and anti-inflammatory activities of these drugs. Thus, the non-anticoagulant activity of heparin has again aroused attention. This review highlights recent advances in the preparation of heparin-derived drugs and clinical research on its non-anticoagulant properties over the past decade, to further the development and utilization of these important drugs., (Copyright © 2022. Published by Elsevier Ltd.)

Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin-core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes., Competing Interests: Declaration of Competing Interest There are no conflict of interest for authors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Non-anticoagulant biological functions of heparin-based drugs have drawn increasing attention. However, the exploration into the non-anticoagulant activities of various low molecular weight heparins was associated with bleeding risks in clinical practice and often led to controversial conclusions due to the structural differences. In this study, we aimed to establish a process to produce a library of heparin derivatives with structural diversity and reduced/abolished anticoagulant activity through the combination of chemical modifications and enzymatic cleavage of heparins. The depolymerization characteristics of various selectively modified heparin derivatives by three heparinases were comprehensively analyzed. The order of periodate treatment and heparinase-I depolymerization was proved to significantly change the structural characteristics of the oligosaccharide products. Finally, among several heparin derivatives that screened in the bleomycin-induced cell apoptosis model, the low molecular weight partially 6-O-/N-desulfated heparins showed the strongest anti-apoptotic activities. This study provided a useful approach for future development of novel heparin-derivative medications., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.

Heparin is a polysaccharide anticoagulant drug isolated from animal tissues. There have been concerns on the safety and security of the heparin supply chain since 2007-8 when a contamination crisis led to its disruption. The current study applies a suite of modern analytical techniques to porcine, bovine and ovine intestinal mucosal heparins. These techniques include structural analysis by nuclear magnetic resonance spectrometry, disaccharide compositional analysis, bottom-up analysis of tetrasaccharides corresponding to heparin's antithrombin III binding site. Chemometric analysis was then applied to understand how these structural differences to predict the animal/tissue source of heparin and to help detect blending of heparins from various sources., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rationale: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts., Methods: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products., Results: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes., Conclusions: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products., (Copyright © 2015 John Wiley & Sons, Ltd.)

Highlights • A method was developed to analyze the complex patterns of enoxaparin/PF4. • The analytical method was developed based on size-exclusion chromatography. • Stability, incubation time and ratios of enoxaparin/FP4 was investigated. • This method monitors the capacity of enoxaparin forming complexes with PF4. Abstract Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. However, heparin-induced thrombocytopenia and thrombosis (HITT) is a serious side effect of heparin therapy, resulting in relatively high risk of amputation and even death. HITT is caused by forming of complexes between heparin and platelet factor 4 (PF4). Enoxaparin, one of the most commonly used low molecular weight heparin (LMWH), were developed in 1980's. The lower molecular weight of enoxaparin reduces the risk of HITT by binding to less PF4. To detect the binding capacity between enoxaparin and PF4 could be an effect way to control this risk before it goes to patients. In this work, a size exclusion chromatography (SEC) method was developed to analyze the patterns of complexes formed between PF4 and enoxaparin. The chromatographic condition was optimized to separate PF4, enoxaparin, ultra-large complexes and small complexes. The linearity and stability of this method were confirmed. The impacts of PF4/enoxaparin mixture ratios and incubation time on the forming complexes were investigated. Four enoxaparin samples were analyzed with this method to verify its practicability. It is a robust, accurate and practicable method, and provides an easy way to monitor the capacity of enoxaparin forming complexes with PF4, suggesting the HITT related quality of enoxaparin. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs), derived from unfractionated heparin (UFH) by chemical or enzymatic degradation, have lower side effects than that of heparin. Enoxaparin, nadroparin, and dalteparin are the most widely used LMWHs. Nadroparin and dalteparin are prepared through nitrous acid degradation followed by a subsequent reduction process, in which specific residue, 2,5-anhydro- d -mannitol (An-Man), is formed at the reducing terminals of generated sugar chains. However, few practicable analytical method was available to analyze An-Man qualitatively and quantitatively. In this work, a HPAEC-PAD-MS method was developed to analyze monosaccharides in heparin and LMWHs, especially An-Man. An ion suppressor is set up between HPAEC and MS to remove the nonvolatile salts from HPAEC and make the elute compatible to MS. Various monosaccharides were separated well with HPAEC. Online MS and MS/MS confirmed all sugar residues in the hydrolysates of heparin samples. The specific residue, An-Man, was only observed in the hydrolysates of LMWHs prepared with nitrous acid degradation and reduction. In addition, major glucosamine and minor arabinose/galactose were observed in all heparin samples. The amounts of these sugar residues were quantitated with PAD, simultaneously. The ratio of glucosamine to An-Man could be used to calculate the molecular weight of LMWHs. The calculated values are comparable to the value measured with size-exclusion chromatography-multiple angle laser scattering detection. Higher the ratio of glucosamine to An-Man higher the molecular weight. The HPAEC-PAD-MS platform is an accurate, precise and efficient way to identify LMWHs by determination of An-Man. It is also an alternative method to detect the MWs of LMWHs having An-Man for quality control purposes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two fractions (A and C) of GAGs from porcine jejunum and duodenum were purified. • A is a heparan sulfate, and C is composed of β- d -GlcA-(1 → 4) and β- d -GalNAc-(1 → 3). • They possessed controllable anti-coagulant activity in vitro. • Additional sulfate modification enhanced the effect. To make full use of the porcine sources and develop better choice of novel GAGs as anti-coagulants, two fractions of GAGs from the porcine jejunum (A) and duodenum (C) have been separated & purified. The products were further sulfated to give B and D in order to test the influence of sulfate pattern on the bioactivity. The results showed that the relative molecular weight range of A was 3000–50,000 (Mw, g/mol), whereas C had an average molecular weight of 75,885 (Mw, g/mol). A was identified as a novel heparan sulfate through enzymatic hydrolysis analysis. C was a chondroitin like polysaccharide mainly composed of β - d -GlcA-(1 → 4) and β - d -GalNAc-(1 → 3). A possessed controllable anti-coagulant activity (7 IU/mg) in vitro. The activity of D almost achieved the same magnitude of A. This study demonstrated the anticoagulant potential of the polysaccharides, providing solid foundation for development of anti-coagulants from porcine intestine. [ABSTRACT FROM AUTHOR]