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ABSTRACT: We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M(w)) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A(2)) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M(w) of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M(w) values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M(w) results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.
Analytical and Bioanalytical Chemistry 09/2011; 401(8):2445-54. · 3.78 Impact Factor
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Qingda Zang,
David A Keire,
Lucinda F Buhse,
Richard D Wood,
Dinesh P Mital,
Syed Haque,
Shankar Srinivasan,
Christine M V Moore, Moheb Nasr,
Ali Al-Hakim,
Michael L Trehy,
William J Welsh
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ABSTRACT: Chemometric analysis of a set of one-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectral data for heparin sodium active pharmaceutical ingredient (API) samples was employed to distinguish USP-grade heparin samples from those containing oversulfated chondroitin sulfate (OSCS) contaminant and/or unacceptable levels of dermatan sulfate (DS) impurity. Three chemometric pattern recognition approaches were implemented: classification and regression tree (CART), artificial neural network (ANN), and support vector machine (SVM). Heparin sodium samples from various manufacturers were analyzed in 2008 and 2009 by 1D (1)H NMR, strong anion-exchange high-performance liquid chromatography, and percent galactosamine in total hexosamine tests. Based on these data, the samples were divided into three groups: Heparin, DS ≤ 1.0% and OSCS = 0%; DS, DS > 1.0% and OSCS = 0%; and OSCS, OSCS > 0% with any content of DS. Three data sets corresponding to different chemical shift regions (1.95-2.20, 3.10-5.70, and 1.95-5.70 ppm) were evaluated. While all three chemometric approaches were able to effectively model the data in the 1.95-2.20 ppm region, SVM was found to substantially outperform CART and ANN for data in the 3.10-5.70 ppm region in terms of classification success rate. A 100% prediction rate was frequently achieved for discrimination between heparin and OSCS samples. The majority of classification errors between heparin and DS involved cases where the DS content was close to the 1.0% DS borderline between the two classes. When these borderline samples were removed, nearly perfect classification results were attained. Satisfactory results were achieved when the resulting models were challenged by test samples containing blends of heparin APIs spiked with non-, partially, or fully oversulfated chondroitin sulfate A, heparan sulfate, or DS at the 1.0%, 5.0%, and 10.0% (w/w) levels. This study demonstrated that the combination of 1D (1)H NMR spectroscopy with multivariate chemometric methods is a nonsubjective, statistics-based approach for heparin quality control and purity assessment that, once standardized, minimizes the need for expert analysts.
Analytical and Bioanalytical Chemistry 06/2011; 401(3):939-55. · 3.78 Impact Factor
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ABSTRACT: Heparin is a naturally produced, heterogeneous compound consisting of variably sulfated and acetylated repeating disaccharide units. The structural complexity of heparin complicates efforts to assess the purity of the compound, especially when differentiating between similar glycosaminoglycans. Recently, heparin sodium contaminated with oversulfated chondroitin sulfate A (OSCS) has been associated with a rapid and acute onset of an anaphylactic reaction. In addition, naturally occurring dermatan sulfate (DS) was found to be present in these and other heparin samples as an impurity due to incomplete purification. The present study was undertaken to determine whether chemometric analysis of these NMR spectral data would be useful for discrimination between USP-grade samples of heparin sodium API and those deemed unacceptable based on their levels of DS, OSCS, or both. Several multivariate chemometric methods for clustering and classification were evaluated; specifically, principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and the k-nearest-neighbor (kNN) method. Data dimension reduction and variable selection techniques, implemented to avoid over-fitting the training set data, markedly improved the performance of the classification models. Under optimal conditions, a perfect classification (100% success rate) was attained on external test sets for the Heparin vs OSCS model. The predictive rates for the Heparin vs DS, Heparin vs [DS+OSCS], and Heparin vs DS vs OSCS models were 89%, 93%, and 90%, respectively. In most cases, misclassifications can be ascribed to the similarity in NMR chemical shifts of heparin and DS. Among the chemometric methods evaluated in this study, we found that the LDA models were superior to the PLS-DA and kNN models for classification. Taken together, the present results demonstrate the utility of chemometric methods when applied in combination with (1)H NMR spectral analysis for evaluating the quality of heparin APIs.
Journal of pharmaceutical and biomedical analysis 04/2011; 54(5):1020-9. · 2.45 Impact Factor
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ABSTRACT: To differentiate heparin samples with varying amounts of dermatan sulfate (DS) impurities and oversulfated chondroitin sulfate (OSCS) contaminants, proton NMR spectral data for heparin sodium active pharmaceutical ingredient samples from different manufacturers were analyzed using multivariate chemometric techniques. A total of 168 samples were divided into three groups: (a) Heparin, [DS] ≤ 1.0% and [OSCS] = 0%; (b) DS, [DS] > 1.0% and [OSCS] = 0%; (c) OSCS, [OSCS] > 0% with any content of DS. The chemometric models were constructed and validated using two well-established methods: soft independent modeling of class analogy (SIMCA) and unequal class modeling (UNEQ). While SIMCA modeling was conducted using the entire set of variables extracted from the NMR spectral data, UNEQ modeling was combined with variable reduction using stepwise linear discriminant analysis to comply with the requirement that the number of samples per class exceed the number of variables in the model by at least 3-fold. Comparison of the results from these two modeling approaches revealed that UNEQ had greater sensitivity (fewer false positives) while SIMCA had greater specificity (fewer false negatives). For Heparin, DS, and OSCS, respectively, the sensitivity was 78% (56/72), 74% (37/50), and 85% (39/46) from SIMCA modeling and 88% (63/72), 90% (45/50), and 91% (42/46) from UNEQ modeling. Importantly, the specificity of both the SIMCA and UNEQ models was 100% (46/46) for Heparin with respect to OSCS; no OSCS-containing sample was misclassified as Heparin. The specificity of the SIMCA model (45/50, or 90%) was superior to that of the UNEQ model (27/50, or 54%) for Heparin with respect to DS samples. However, the overall prediction ability of the UNEQ model (85%) was notably better than that of the SIMCA model (76%) for the Heparin vs DS vs OSCS classes. The models were challenged with blends of heparin spiked with nonsulfated, partially sulfated, or fully oversulfated chondroitin sulfate A, dermatan sulfate, or heparan sulfate at the 1.0, 5.0, and 10.0 wt % levels. The results from the present study indicate that the combination of (1)H NMR spectral data and class modeling techniques (viz., SIMCA and UNEQ) represents a promising strategy for assessing the quality of commercial heparin samples with respect to impurities and contaminants. The methodologies show utility for applications beyond heparin to other complex products.
Analytical Chemistry 02/2011; 83(3):1030-9. · 5.86 Impact Factor
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ABSTRACT: During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.
Analytical and Bioanalytical Chemistry 01/2011; 399(2):581-91. · 3.78 Impact Factor
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ABSTRACT: Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.
Analytical Chemistry 11/2010; 82(23):9865-70. · 5.86 Impact Factor
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ABSTRACT: Heparin, a widely used anticoagulant primarily extracted from animal sources, contains varying amounts of galactosamine impurities. Currently, the United States Pharmacopeia (USP) monograph for heparin purity specifies that the weight percent of galactosamine (%Gal) may not exceed 1%. In the present study, multivariate regression (MVR) analysis of (1)H NMR spectral data obtained from heparin samples was employed to build quantitative models for the prediction of %Gal. MVR analysis was conducted using four separate methods: multiple linear regression, ridge regression, partial least squares regression, and support vector regression (SVR). Genetic algorithms and stepwise selection methods were applied for variable selection. In each case, two separate prediction models were constructed: a global model based on dataset A which contained the full range (0-10%) of galactosamine in the samples and a local model based on the subset dataset B for which the galactosamine level (0-2%) spanned the 1% USP limit. All four regression methods performed equally well for dataset A with low prediction errors under optimal conditions, whereas SVR was clearly superior among the four methods for dataset B. The results from this study show that (1)H NMR spectroscopy, already a USP requirement for the screening of contaminants in heparin, may offer utility as a rapid method for quantitative determination of %Gal in heparin samples when used in conjunction with MVR approaches.
Analytical and Bioanalytical Chemistry 10/2010; 399(2):635-49. · 3.78 Impact Factor
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Takashi Kei Kishimoto,
Karthik Viswanathan,
Tanmoy Ganguly,
Subbiah Elankumaran,
Sean Smith,
Kevin Pelzer,
Jonathan C Lansing,
Nammalwar Sriranganathan,
Ganlin Zhao,
Zoya Galcheva-Gargova, [......],
Mark Whary,
James Fox, Moheb Nasr,
Gerald J Dal Pan,
Zachary Shriver,
Robert S Langer,
Ganesh Venkataraman,
K Frank Austen,
Janet Woodcock,
Ram Sasisekharan
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ABSTRACT: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany.
Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine.
The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine.
Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.
New England Journal of Medicine 07/2008; 358(23):2457-67. · 53.30 Impact Factor
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Marco Guerrini,
Daniela Beccati,
Zachary Shriver,
Annamaria Naggi,
Karthik Viswanathan,
Antonella Bisio,
Ishan Capila,
Jonathan C Lansing,
Sara Guglieri,
Blair Fraser, [......],
Luke Robinson,
Lucinda Buhse, Moheb Nasr,
Janet Woodcock,
Robert Langer,
Ganesh Venkataraman,
Robert J Linhardt,
Benito Casu,
Giangiacomo Torri,
Ram Sasisekharan
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ABSTRACT: Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.
Nature Biotechnology 06/2008; 26(6):669-75. · 29.50 Impact Factor
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Nakissa Sadrieh,
James Brower,
Lawrence Yu,
William Doub,
Arthur Straughn,
Stella Machado,
Frank Pelsor,
Emmanuelle Saint Martin,
Terry Moore,
John Reepmeyer,
Duckhee Toler,
Agnes Nguyenpho,
Rosemary Roberts,
Donald J Schuirmann, Moheb Nasr,
Lucinda Buhse
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ABSTRACT: These studies evaluated the ability of common household food and drink products to mask the bitter taste of three selected anti-terrorism drugs.
Three anti-terrorism drugs (doxycycline, ciprofloxacin hydrochloride, and potassium iodide) were mixed with a variety of common household food and drinks, and healthy adult volunteers evaluated the resulting taste and aftertaste. In parallel, the ASTREE Electronic Tongue was used to evaluate taste combinations. Stability of the mixtures over time was monitored, as was the dosage uniformity across preparations.
Foods and drinks were identified that satisfactorily masked the bitter flavor of each drug. Dose uniformity and stability were also acceptable over the range studied, although some combinations were significantly less stable than others. The electronic tongue was able to differentiate between tastes, but ranked masking agents in a different order than human volunteers.
Doxycycline, potassium iodide, and ciprofloxacin, which are stockpiled in solid tablet form, can conveniently be prepared into more palatable formulations, using common household foods and drinks. The electronic tongue can be used to perform an initial screening for palatability.
Pharmaceutical Research 11/2005; 22(10):1747-56. · 4.09 Impact Factor
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ABSTRACT: Heparin is a naturally produced, heterogeneous compound consisting of variably sulfated and acetylated repeating disaccharide units. The structural complexity of heparin complicates efforts to assess the purity of the compound, especially when differentiating between similar glycosaminoglycans. Recently, heparin sodium contaminated with oversulfated chondroitin sulfate A (OSCS) has been associated with a rapid and acute onset of an anaphylactic reaction. In addition, naturally occurring dermatan sulfate (DS) was found to be present in these and other heparin samples as an impurity due to incomplete purification. The present study was undertaken to determine whether chemometric analysis of these NMR spectral data would be useful for discrimination between USP-grade samples of heparin sodium API and those deemed unacceptable based on their levels of DS, OSCS, or both. Several multivariate chemometric methods for clustering and classification were evaluated; specifically, principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and the k-nearest-neighbor (kNN) method. Data dimension reduction and variable selection techniques, implemented to avoid over-fitting the training set data, markedly improved the performance of the classification models. Under optimal conditions, a perfect classification (100% success rate) was attained on external test sets for the Heparin vs OSCS model. The predictive rates for the Heparin vs DS, Heparin vs [DS + OSCS], and Heparin vs DS vs OSCS models were 89%, 93%, and 90%, respectively. In most cases, misclassifications can be ascribed to the similarity in NMR chemical shifts of heparin and DS. Among the chemometric methods evaluated in this study, we found that the LDA models were superior to the PLS-DA and kNN models for classification. Taken together, the present results demonstrate the utility of chemometric methods when applied in combination with 1H NMR spectral analysis for evaluating the quality of heparin APIs.
Journal of Pharmaceutical and Biomedical Analysis.
