Molecular Profiling and Weight Determination of Heparins and Depolymerized Heparins

Ahsan, Ahmad; Jeske, Walter; Hoppensteadt, Debra; Lormeau, J C; Wolf, Helmut; Fareed, Jawed

doi:10.1002/jps.2600840612

Cited by 40 publications

(31 citation statements)

References 6 publications

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“…8,9 The polydispersity (the ratio of weight averaged to number averaged molecular weight) of pharmaceutical heparin is 1.1–1.6. 8 The chains making up polydisperse pharmaceutical grade heparin range from 5,000 to over 40,000 Da 3 and contain a significant level of sequence heterogeneity. Heparin is composed of a major (75–95%) trisulfated disaccharide repeating unit (Fig.…”

Section: Heparin Structure and Activitiesmentioning

confidence: 99%

Lessons learned from the contamination of heparin

2009

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Heparin is unique as one of the oldest drugs currently still in widespread clinical use as an anticoagulant, a natural product, one of the first biopolymeric drugs, and one of the few carbohydrate drugs. Recently, certain batches of heparin have been associated with anaphylactoid-type reactions, some leading to hypotension and death. These reactions were traced to contamination with a semi-synthetic oversulfated chondroitin sulfate (OSCS). This Highlight reviews the heparin contamination crisis, its resolution, and the lessons learned. Pharmaceutical scientists now must consider dozens of natural and synthetic heparinoids as potential heparin contaminants. Effective assays, which can detect both known and unknown contaminants, are required to monitor the quality of heparin. Safer and better-regulated processes are needed for heparin production.

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Section: Heparin Structure and Activitiesmentioning

confidence: 99%

Lessons learned from the contamination of heparin

2009

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“…Heparin chains are polydisperse (molecular weight: 5,000 – 40,000) and contain significant sequence heterogeneity (Ahsan et al, 1995). Fully sulfated heparin chains are composed of uniform, repeating sequences of trisulfated disaccharides whereas heparin chains having intermediate level of sulfation (2.5 sulfo groups per disaccharide) are composed of long segments of fully sulfated sequences with intervening undersulfated domains (Fig.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

Baik

Gasimli

Yang

et al. 2012

Metabolic Engineering

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Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary.

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“…Individual laboratories have produced such standards on a small scale and characterized them by viscosity measurements [22], light scattering [23], or a combination of both [24]. The production of MW markers of this type for unfractionated heparin on a large scale is a difficult task.…”

Section: Introductionmentioning

confidence: 99%