Oversulfated Chondroitin Sulfate In Heparin Research Articles

Recent developments in sensing of oversulfated chondroitin sulfate in heparin. A review.

Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could trigger adverse reactions. Chromatography-, electrophoresis-, electrochemistry-, and spectroscopy-related techniques are currently available for accurate and precise analysis of a trace amount of OSCS in heparin. Recently, emerging studies focus on developing colorimetric and fluorescent probes to monitor OSCS containments in heparin. Therefore, this current review aims to describe the sensing principle and procedure of the reported probes that are sensitive and selective toward OSCS in heparin without the interferences of other sulfated glycosaminoglycans. The reported OSCS-specific probes are comprehensively discussed according to the recognition elements of OSCS, including coralyne, AG73 peptides, positively charged tetraphenylethene derivatives, polythiophene polymer, and poly-L-lysine, protamine, superpositively charged green fluorescent proteins, and poly (diallyldimethylammonium chloride). The sensing of OSCS in heparin is generally achieved using, (i) the specific affinity of the recognition element with OSCS and heparin, (ii) heparinase-mediated hydrolysis of heparin, and (iii) OSCS-induced inhibition of heparinase activity. Additionally, coralyne-based DNA probes can detect OSCS in heparin in the presence of Ca2+ ions without the assistance of heparinase. This review will pave the way to design another sensing probe towards other sulfated contaminants, like dermatan sulfate.

Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe

The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R2=0.99) in aqueous solutions, 2.98nM (R2=0.98) in 1% serum samples, and 3.43nM (R2=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.

Molecular Beacon-Based Fluorescent Assay for Specific Detection of Oversulfated Chondroitin Sulfate Contaminants in Heparin without Enzyme Treatment.

Oversulfated chondroitin sulfate (OSCS) is a harmful contaminant in the pharmaceutical heparin. The development of a rapid, convenient, sensitive, and selective method is required for routine analysis of OSCS in pharmaceutical heparin. Here we report a simple, rapid, sensitive, and enzyme-free method for detecting OSCS in heparin based on the competitive binding between OSCS and the adenosine-repeated molecular beacon (MB) stem to coralyne in the presence of Ca(2+) ions. The MB (A8-MB-A8) contains a 22-mer loop, a stem of a pair of 8-mer adenosine (A) bases, a fluorophore unit at the 5'-end, and a quencher at the 3'-end. The presence of coralyne promotes these A-A mismatches to form a hairpin-shaped MB. However, this kind of MB is incapable of differentiating between heparin and OSCS because they both exhibit strong electrostatic attraction with coralyne. This study found that while Ca(2+) ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS. Thus, in the presence of Ca(2+) ions, OSCS can remove coralyne from the MB stem, initiating fluorescence of the MB. Under optimal conditions (10 nM A8-MB-A8, 800 nM coralyne, and 0.5 mM Ca(2+) ions), the proposed system can detect 0.01% w/w OSCS in heparin in under 5 min without enzyme treatment. This study also validates the practicality of the proposed system to determine 0.01% w/w OSCS in the pharmaceutical heparin.

A Nanosensor for Ultrasensitive Detection of Oversulfated Chondroitin Sulfate Contaminant in Heparin

Heparin has been extensively used as an anticoagulant for the last eight decades. Recently, the administration of a contaminated batch of heparin caused 149 deaths in several countries including USA, Germany, and Japan. The contaminant responsible for the adverse effects was identified as oversulfated chondroitin sulfate (OSCS). Here, we report a rapid, ultrasensitive method of detecting OSCS in heparin using a nanometal surface energy transfer (NSET) based gold-heparin-dye nanosensor. The sensor is an excellent substrate for heparitinase enzyme, as evidenced by ~70% recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, as the enzyme is inhibited by the contaminant. The newly designed nanosensor can detect as low as 1 × 10(-9) % (w/w) OSCS making it the most sensitive tool to date for the detection of trace amounts of OSCS in pharmaceutical heparins.

High-Throughput Differentiation of Heparin from Other Glycosaminoglycans by Pyrolysis Mass Spectrometry

Sensors with high chemical specificity and enhanced sample throughput are vital to screening food products and medical devices for chemical or biochemical contaminants that may pose a threat to public health. For example, the rapid detection of oversulfated chondroitin sulfate (OSCS) in heparin could prevent reoccurrence of heparin adulteration that caused hundreds of severe adverse events including deaths worldwide in 2007-2008. Here, rapid pyrolysis is integrated with direct analysis in real time (DART) mass spectrometry to rapidly screen major glycosaminoglycans, including heparin, chondroitin sulfate A, dermatan sulfate, and OSCS. The results demonstrate that, compared to traditional liquid chromatography-based analyses, pyrolysis mass spectrometry achieved at least 250-fold higher sample throughput and was compatible with samples volume-limited to about 300 nL. Pyrolysis yielded an abundance of fragment ions (e.g., 150 different m/z species), many of which were specific to the parent compound. Using multivariate and statistical data analysis models, these data enabled facile differentiation of the glycosaminoglycans with high throughput. After method development was completed, authentically contaminated samples obtained during the heparin crisis by the FDA were analyzed in a blinded manner for OSCS contamination. The lower limit of differentiation and detection were 0.1% (w/w) OSCS in heparin and 100 ng/μL (20 ng) OSCS in water, respectively. For quantitative purposes the linear dynamic range spanned approximately 3 orders of magnitude. Moreover, this chemical readout was successfully employed to find clues in the manufacturing history of the heparin samples that can be used for surveillance purposes. The presented technology and data analysis protocols are anticipated to be readily adaptable to other chemical and biochemical agents and volume-limited samples.

Competitive Inhibition of Heparinase by Persulfonated Glycosaminoglycans: A Tool to Detect Heparin Contamination

Heparin and the low molecular weight heparins are extensively used as medicinal products to prevent and treat the formation of venous and arterial thrombi. In early 2008, administration of some heparin lots was associated with the advent of severe adverse effects, indicative of an anaphylactoid-like response. Application of orthogonal analytical tools enabled detection and identification of the contaminant as oversulfated chondroitin sulfate (OSCS) was reported in our earlier report. Herein, we investigate whether enzymatic depolymerization using the bacterially derived heparinases, given the structural understanding of their substrate specificity, can be used to identify the presence of OSCS in heparin. We also extend this analysis to examine the effect of other persulfonated glycosaminoglycans (GAGs) on the action of the heparinases. We find that all persulfonated GAGs examined were effective inhibitors of heparinase I, with IC(50) values ranging from approximately 0.5-2 μg/mL. Finally, using this biochemical understanding, we develop a rapid, simple assay to assess the purity of heparin using heparinase digestion followed by size-exclusion HPLC analysis to identify and quantify digestion products. In the context of the assay, we demonstrate that less than 0.1% (w/w) of OSCS (and other persulfonated polysaccharides) can routinely be detected in heparin.

Highly Sensitive Potentiometric Strip Test for Detecting High Charge Density Impurities in Heparin

Contamination of heparin with oversulfated chondroitin sulfate (OSCS) became a matter of grave concern in the medical field after many fatal responses to OSCS tainted heparin products occurred during the 2007-2008 period. Even though standard lab-based analytical techniques such as nuclear magnetic resonance (NMR) and strong anion-exchange high performance liquid chromatography (SAX-HPLC) have proven useful for monitoring the OSCS content in heparin products, an easy-to-use, quick, portable, and cost-efficient method is still needed for on-site monitoring during and after the heparin production. In this report, a disposable strip-type electrochemical polyion sensor is described for detection of low levels of OSCS contamination in heparin. A magnetic actuator is incorporated into this simple electrode-based microfluidic device in order to create the mixing effect necessary to achieve equilibrium potential changes of the sensor within a microfluidic channel. The planar membrane electrode detector within the sample channel is prepared with a tridodecylmethylammonium chloride (TDMAC)-doped poly(vinyl chloride) (PVC) membrane essentially equivalent to previously reported polyanion-sensitive electrodes. When the concentration of heparin applied to the single-use strip device is 57 mg/mL (in only 20 μL of sample), the same concentration recommended in the NMR analysis protocol for detecting OSCS in heparin, the detection limit is 0.005 wt % of OSCS, which is ca. 20 times lower than the reported detection limit of the NMR method.

Simple fluorescence assay for quantification of OSCS in heparin

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ ((em)) 330nm, λ ((ex)) 510nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with (1)H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.

Determination of oversulfated chondroitin sulfate and dermatan sulfate impurities in heparin by capillary electrophoresis

Recently, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. The United States Pharmacopeia (USP) and European Pharmacopeia (Eur.Ph.) announced an immediate revision of their monographs for heparin sodium by adding two US Food and Drugs Administration-recommended tests for OSCS based on nuclear magnetic resonance and capillary electrophoresis (CE). However, the proposed CE method provides only partial separation of the OSCS contaminant from heparin, thereby hindering appropriate impurity profiling. Here we present an improved CE method that is especially useful for the reliable quantification of OSCS in heparin samples, but also allows determination of the common impurity dermatan sulfate (DS). Parameters such as type and concentration of background electrolyte, capillary temperature, sample concentration and injection volume were investigated and optimized. Enhancement of the OSCS–heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. High currents and excessive Joule heating were prevented by employing fused-silica capillaries with an internal diameter of 25 μm. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits injection of relatively high heparin concentrations (up to 50 mg/ml) and large sample volumes (up to 5% of the capillary volume) allowing OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method is shown to be repeatable and linear ( R 2 > 0.99) for OSCS, heparin and DS. CE analyses of OSCS-contaminated heparin samples and different heparin standards further demonstrate the utility of the method.

Analysis of heparin sodium by SAX/HPLC for contaminants and impurities

A chromatographic method was developed for the detection and quantification of the contaminant oversulfated chondroitin sulfate (OSCS) and the impurity dermatan sulfate in heparin active pharmaceutical ingredient (API). The HPLC analysis of heparin is carried out using a polymer-based strong anion exchange (SAX) column with gradient elution from 0.125M sodium chloride to 2.5M sodium chloride buffered mobile phase. The limit of detection (LOD) and limit of quantitation (LOQ) for the contaminant OSCS in heparin were determined to be 0.03% and 0.1%, respectively. The LOD and LOQ for dermatan sulfate, an impurity in heparin sulfate, were determined to be 0.1% and 0.8%, respectively. This manuscript is not a policy document and is not intended to replace either of the methods (capillary electrophoresis and NMR) currently required by the FDA.