Bioengineering of Human Heparin: A Next Generation Anti‐coagulant

Abstract

Heparin used clinically is manufactured by extraction from porcine and bovine tissues. Recently, contaminating oversulfated chondroitin sulfates found in batches of porcine heparin led to the deaths of approximately 100 people in the US, which was caused by an uncontrolled anaphylactoid‐type response. This has led researchers to explore alternative methods of producing heparin including chemical synthesis, chemoenzymatic synthesis and metabolic engineering of cells to express high levels of the enzymes involved in heparin biosynthesis. However, these approaches are yet to produce heparins with anticoagulant activity similar to heparin used in the clinic. The aim of this research was to recombinantly express serglycin in human cells as an alternative approach to producing anticoagulant heparin.The serglycin recombinantly expressed in human embryonic kidney (HEK‐293) cells was decorated with heparin, heparan sulfate and chondroitin sulfate. The disaccharide composition of the recombinantly expressed heparin was analysed by fluorophore assisted carbohydrate electrophoresis and contained 15% tri‐sulfated disaccharides as well as 40% di‐sulfated, 25% monosulfated and 20% unsulfated disaccharides. The disaccharide composition of low molecular weight heparin was also analysed and contained a higher proportion of sulfated disaccharides than the recombinantly expressed heparin being 40% trisulfated, 45% disulfated, 10% monosulfated and 5% unsulfated disaccharides.The activated partial clotting time assay is used clinically to monitor heparin levels in patients after its administration and was used in this study to determine the anticoagulant activity of the recombinant human heparin and to compare this to the activity of low molecular weight porcine heparin. In this assay porcine heparin was approximately 20 times more potent in its anticoagulant activity than the recombinant human heparin on a weight basis.These data demonstrate for the first time the successful production of human heparin from cells transfected with the human serglycin gene. This is a significant step towards alternative sources of heparin that has distinct advantages for patient safety due to the use of production methods with a lower inherent risk of contamination. Human cell‐derived heparin will also provide an alternative source to animal‐derived heparin that will enable its use by patients who, due to religious beliefs and/or dietary choices, have so far been excluded from heparin treatment.Support or Funding InformationFunding provided by the Australian Research Council.