(586k) Chemoenzymatic Synthesis of Heparin for a Safer Bioengineered Alternative

Heparin, the first biopolymeric drug, possesses a wide range of structural heterogeneity owing to its biosynthesis. Its diverse fine structure is further complicated by an animal tissue-based recovery, leading to considerable structural differences within commercial heparin active pharmaceutical ingredients (APIs). Serious concerns about control of livestock, the primary source of heparin, have been raised since 1990s following a series of incidents involving Bovine spongiform encephalopathy, viral infections and prion contamination. Lack of quality control during initial recovery stages led to adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS), resulting in an international crisis in 2008 associated with 100 deaths in US.

The inherent problems with the animal tissue-based heparin production have motivated us to develop a commercially feasible chemoenzymatic heparin preparation process.  This is based on bacterial fermentation of E. coli K5 to generate a capsular polysaccharide heparin precursor, that is then chemically N-deacetylated and N-sulfonated.  A series of six recombinant enzymes, derived from heparin biosynthetic pathway and expressed in E. coli, are then used to epimerize uronic acid residues and sulfonate the C2, C3 and C6 positions. These modifications are carried out in tandem with sensitive analytical techniques and bioassays to result in a series of controlled structural changes. Bioengineered heparin consists of 86% trisulfated disaccharide resembling the highly sulfated structure of heparin. The formation of 9.4% lyase resistant tetrasaccharides upon digestion with heparin lyase 2 is consistent with the presence of antithrombin binding pentasaccharide regions in the range of USP heparins (3.9-6.7%). 1D-¹H NMR and HMQC techniques were employed to elucidate presence of 3-O-sulfo and I_2S peak in anomeric regions. Peaks corresponding to critical features in the IdoA and GlcN residues, including 2-O-sulfo, N-sulfo, N-acetyl, 3-O-sulfo, and 6-O-sulfo, have been fully assigned by ¹H-NMR and HMQC spectra. Investigation of in-vitro anticoagulant activity confirms the biological activity of chemoenzymatically derived heparin with an anti-factor Xa activity of 205.8 U/mg and an anti-factor IIa activity of 225.2 U/mg (anti-factor Xa:anti-factor IIa ratio of 0.91) complying with USP requirements. These analyses showcase generic equivalence of bioengineered heparin to animal derived heparin API. A more robust process based on immobilized enzymes, enhancing enzymatic stability and increased reusability, is under development and will be employed to generate bioengineered heparin for in-vivo studies.