Validation of quantitative polymerase chain reaction methodology for monitoring DNA as a surrogate marker for species material contamination in porcine heparin.
Heparin is a widely used intravenous anticoagulant comprising of a very complex mixture of glycosaminoglycan chains, mainly derived from porcine intestinal mucosa. The species of origin and the absence of contaminants from other species are important determinants of the different physicochemical characteristics of heparin. They also determine the potential for introducing infectious and adventitious agents into heparin batches destined for medicinal use. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches, including those used for the manufacture of enoxaparin sodium. Here we further demonstrate that the assessment of the DNA content in crude heparin is a good surrogate marker of contamination at the mucosa level. After spiking porcine mucosa with ovine mucosa and processing this material to form crude heparin, we were able to observe similar ratios of species-specific DNA in both the starting and end products. Experiments performed with 3,000 and 1,500 ppm contamination found these concentrations to be well above the detection limit for our assay of heparin batches. Additionally this Q-PCR method can be used to detect contamination in mucosa, thus providing a tool capable of monitoring for contaminants throughout the crude heparin manufacturing process. Q-PCR analysis of industrial crude heparin samples has confirmed over time the value of this method to assess the pure porcine origin of heparin.
Process Development Biotechnology Division, Sanofi, Vitry-sur-Seine, France.
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C
Auguste
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S
Dereux
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M
Rousset
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P
Anger
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D004247:DNA / Q000235:genetics*; D004340:Drug Contamination / Q000517:prevention & control*; D006493:Heparin / Q000737:chemistry* / Q000592:standards; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012756:Sheep / Q000235:genetics*; D013045:Species Specificity; D013552:Swine / Q000235:genetics*
D015415:Biomarkers; D006493:Heparin; D004247:DNA