Heparin is a widely used anticoagulant and antithrombotic agent. Recently, a contaminant, oversulfated chondroitin sulfate (OSCS), was discovered within heparin preparations. The presence of OSCS within heparin likely led to clinical manifestations, most prevalently, hypotension and abdominal pain leading to the deaths of several dozens of patients. Given the biological effects of OSCS, one continuing item of concern is the ability for existing methods to identify other persulfonated polysaccharide compounds that would also have anticoagulant activity and would likely elicit a similar activation of the contact system. To complete a more extensive analysis of the ability for NMR and capillary electrophoresis (CE) to capture a broader array of potential contaminants within heparin, we completed a systematic study of NMR, both mono- and bidimensional, and CE to detect both various components of sidestream heparin and their persulfonated derivatives. We show that given the complexity of heparin samples, and the requirement to ensure their purity and safety, use of orthogonal analytical techniques is effective at detecting an array of potential contaminants that could be present.

https://www.pnas.org/content/pnas/106/40/16956.full.pdf

Orthogonal analytical approaches to detect potential contaminants in heparin

Glycosaminoglycans (GAG) such as hyaluronan (HA) or chondroitin/dermatan sulfate (CS/DS) occur in many connective tissues, for instance, in bone, cartilage and skin. Due to their significant water-binding capacity, GAG are essential for the biomechanical properties of these tissues. However, there is also increasing evidence that the sulfation of GAG does not occur at random, but a "sulfation code" exists that mediates the physiological functions of GAG. Thus, the biological properties of these biomacromolecules are strongly influenced by the degree of sulfation (ds) and the sulfate group distribution along the polymer. Therefore, certain GAG might also have interesting pharmacological properties. It is, thus, commonly accepted that GAG represent promising biomaterials in the field of tissue engineering as well as to design new bioactive materials for tissue repair and reconstruction. In this review we will focus on chemically sulfated GAG and provide a survey of these compounds on four different levels. First, we will provide an overview on chemical functionalization strategies of naturally occurring HA and CS/DS with special emphasis on regioselective methods to introduce a defined number of sulfate residues into the carbohydrate backbone. Second, chemical and biochemical methods to characterize the synthesized compounds will be introduced with the focus on methods based on nuclear magnetic resonance (NMR) and mass spectrometry (MS). In the third part, we will discuss the interaction of natural and chemically sulfated GAG with proteins and other biomolecules with regulatory functions. Additionally, biological consequences of these interactions regarding healing processes of skin and bone will be presented by discussing selected cell culture experiments. Finally, in vivo effects of GAG as components of artificial extracellular matrices will be discussed.

Sulfated glycosaminoglycans as promising artificial extracellular matrix components to improve the regeneration of tissues.

Biologically active ingredients and excipients are the essentials of a drug formulation, such as a tablet, dragee, solution, etc. Quality control of such substances thus plays a pivotal role in the production process of pharmaceutical drugs. Since these agents often exhibit complex structures, consist of multiple components, or lack of a chromophore, traditional means of characterization are often not feasible. Furthermore, substances of small molecular weight or strong polar character generally exhibit poor chromatographic properties, thus, conventional procedures such as high-performance liquid chromatography are often not applicable. Instead, quantitative nuclear magnetic resonance (qNMR) spectroscopy has emerged as an alternative or orthogonal method in drug analysis. In this review, we elaborate on the application of qNMR to three important classes of biological substances, namely polysaccharides, amino acids, and lipids, and demonstrate the benefits of this modern tool in contrast to traditional techniques.

Quantitative NMR spectroscopy of biologically active substances and excipients

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.

/pdf/electrophoresis-for-the-analysis-of-heparin-purity-and-3iyx96cjci.pdf

Electrophoresis for the analysis of heparin purity and quality.

Background: The contaminant isolated from contaminated heparin was oversulfated chondroitin sulfate (OSCS). Other possible contaminants should be evaluated. Methods: Contaminants were isolated from...

https://journals.sagepub.com/doi/pdf/10.1177/1076029610392214

Review: contaminants in heparin: review of the literature, molecular profiling, and clinical implications.

The detection of a contamination of heparin with oversulphated chondroitin sulphate (OSCS) was first analysed in an unfractionated heparin batch supplied to the US API-market in April 2006. OSCS is a semi-synthetic derivative of the natural occuring glycosaminoglycan chondroitin sulphate. Moreover some spectroscopic characteristics of the substance overlap with those of heparin, so that the infrared (IR) spectra are visually difficult to distinguish whereas (1)H-NMR (Nuclear Magnetic Resonance) spectroscopy or capillary electrophoresis (CE) provides identification by a simple visual inspection of either the spectrum or the electropherogram respectively. However, applying special tools of Multivariate Data Analysis (MVA) to the IR spectra an identification of the contaminated samples is possible. In detail a rapid Attenuation Total Reflectance-Infrared (ATR-IR) measurement was selected, which does not require any sample preparation. The result (contaminated or not contaminated) is predicted within a few minutes. A method transfer to mobile ATR-IR spectrometers seems to be possible. The analysis is based on the fact that the fingerprint of the OSCS IR spectrum (1st derivative) complies with a theoretically calculated principal component in the MVA.

Prediction of the oversulphated chondroitin sulphate contamination of unfractionated heparin by ATR-IR spectrophotometry.

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Prediction of the oversulphated chondroitin sulphate contamination of unfractionated heparin by ATR-IR spectrophotometry.