Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

Li, Guoyun; Yang, Bo; Li, Lingyun; Zhang, Fuming; Xue, Changhu; Linhardt, Robert J.

doi:10.1016/j.ab.2014.02.033

Cited by 39 publications

(31 citation statements)

References 32 publications

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“…26 This enzyme is known to show the least selectivity among the 3 Flavobacterial heparin lyases 21,27,28 cleaving both highly sulfated and undersulfated domains within heparin. 27,28 Despite its broad specificity, certain tetrasaccharides are resistant to heparin lyase II digestion.…”

Section: Resultsmentioning

confidence: 99%

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Chen

Lei

Agyekum

et al. 2017

Journal of Pharmaceutical Sciences

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Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin’s anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.

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Section: Resultsmentioning

confidence: 99%

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Chen

Lei

Agyekum

et al. 2017

Journal of Pharmaceutical Sciences

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“…The bound AT undergoes a conformational change that enhances its ability to inactivate thrombin, responsible for conversion of soluble fibrinogen into an insoluble fibrin clot, leading to observed anticoagulant activity of heparins. The representative pentasaccharide binding sequence of heparin is a collection of sequences and can vary depending upon the source (Li et al, 2014). When heparin is exhaustively treated with heparin lyase 2, in addition to the disaccharides formed, some lyase resistant tetrasaccharides are observed due to the presence of 3- O -sulfo containing glucosamine residues (Li et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

“…The representative pentasaccharide binding sequence of heparin is a collection of sequences and can vary depending upon the source (Li et al, 2014). When heparin is exhaustively treated with heparin lyase 2, in addition to the disaccharides formed, some lyase resistant tetrasaccharides are observed due to the presence of 3- O -sulfo containing glucosamine residues (Li et al, 2014). Their molecular ratio provides a fingerprint of the heparin from which they were derived as well as an insight into the structural diversity of the AT- binding pentasaccharide sequence within heparin.…”

Section: Resultsmentioning

confidence: 99%

“…Their molecular ratio provides a fingerprint of the heparin from which they were derived as well as an insight into the structural diversity of the AT- binding pentasaccharide sequence within heparin. A positive correlation between the 3- O -sulfo containing glucosamine content to in vitro biological activity of USP porcine heparins has been observed (Li et al, 2014). We analyzed seven different USP heparins obtained from commercial sources, as described previously (Zhang et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

“…For tetrasaccharide analysis, 40 mU of heparin lyase 2 in 20 μL of 25 mM Tris, 500 mM NaCl, 300 mM imidazole buffer (pH 7.4) was added to 50-100 μg heparin sample in 100 μL of distilled water and incubated at 35 °C for 10 h. The resulting product was freeze-dried for further LC-MS analysis (Li et al, 2014). …”

Section: Methodsmentioning

confidence: 99%

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Combinatorial one-pot chemoenzymatic synthesis of heparin

Bhaskar

et al. 2015

Carbohydrate Polymers

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Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/ N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal- derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin’s characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

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Optimization of bioprocess conditions improves production of a CHO cell‐derived, bioengineered heparin

Baik

Dahodwala

Oduah

et al. 2015

Biotechnology Journal

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Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S® cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ~100-fold and the heparin/heparan sulfate yield ~10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ~two-fold increase in integrated viable cell density and 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ~90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

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Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

Cited by 39 publications

References 32 publications

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Structural Analysis of Heparin-Derived 3- O -Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Combinatorial one-pot chemoenzymatic synthesis of heparin

Optimization of bioprocess conditions improves production of a CHO cell‐derived, bioengineered heparin

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