Control of the heparosan N-deacetylation leads to an improved bioengineered heparin

Abstract

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.

Fig. 1

The scheme for producing bioengineered anticoagulant heparin from E. coli K5 heparosan

Fig. 2

a ¹H NMR of N-sulfo, N-acetyl heparosan from 3 h N-deacetylated K5 heparosan. b Remaining N-acetyl content as a function of N-deacetylation reaction time. Error bar represents the standard deviation

Fig. 3

a Size exclusion chromatogram of K5 heparosan (top) and N-deacetylated K5 heparosan (bottom). M_N, M_W, and PDI calculated from the chromatogram for each sample are annotated on the figure. b Polyacrylamide gel electrophoresis gel of bovine lung heparin ladder (lane 1), bioengineered heparin (lane 2), and pharmaceutical porcine intestinal heparin (lane 3). M_N, M_W, and PDI calculated from the gel for each sample are annotated on the figure

Fig. 4

¹H NMR of bioengineered heparin (a) and commercial heparin sample (b)

Fig. 5

Liquid chromatography–mass spectrometry of disaccharides the most commonly found in heparan sulfate/heparin. a Extracted ion chromatogram of the bioengineered heparin; b extracted ion chromatogram of the commercial heparin (0S=ΔUA-GlcNAc, NS=ΔUA-GlcNS, 6S=ΔUA-GlcNAc6S, 2S=ΔUA2S-GlcNAc, NS6S=ΔUA-GlcNS6S, NS2S=ΔUA2S-GlcNS, 2S6S=ΔUA2S-GlcNAc6S, TriS=ΔUA2S-GlcNS6S)

Fig. 6

Activated partial thromboplastin time assay of bioengineered heparin and pharmaceutical porcine intestinal heparin. The basal activated partial thromboplastin time was 30.5±0.25 s, data represents mean±SD, n= 3