Background: Although most biologics are produced using recombinant technologies, heparin persists as a product purified from animal tissues. A cell based system for production of heparin would eliminate risk of supply shortage and contamination. While heparin is produced uniquely by mast cells, all mammalian cells produce heparan sulfate, a closely related and non-anticoagulant polysaccharide, in a shared biosynthetic pathway. Production of recombinant heparin in mammalian cells requires engineering the heparan sulfate biosynthetic pathway, which consists of more than 20 biosynthetic enzymes, to produce material with anticoagulant activity.

Aims: This work is focused on engineering mammalian cell lines and bioprocess methods to produce recombinant heparin.

Methods: The heparan sulfate biosynthetic pathway of mastocytoma cells was genetically engineered to alter the expression of heparan sulfate sulfotransferases. The resulting cell lines were screened for production of anti-Xa activity. Heparan sulfate production from a candidate cell line in chemically defined medium was tested. The recombinant product was characterized structurally and in clotting and anti-protease assays.

Results: Overexpression of sulfotransferases in engineered mastocytoma cells produced heparan sulfate with anti-Xa (297 U/mg) and anti-IIa (311 U/mg) activity exceeding the USP requirement for unfractionated heparin despite having lower sulfate content. APTT activity was similar to unfractionated heparin. Chain length was longer than unfractionated heparin.

Conclusions: Genetic manipulation of the biosynthetic pathway yielded heparan sulfate with anticoagulant potency. These results demonstrate the feasibility of producing a substitute for unfractionated heparin from recombinant cell culture.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/recombinant-heparin-an-old-drug-for-the-modern-world/