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Heparin, a highly sulfated glycosaminoglycan (GAG), is used extensively as an anticoagulant [1]. Heparin, and its analogues, are used during surgery and dialysis, and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Administered parenterally, often continuously due to its short half-life, over 0.5 billion doses are required per year. Currently obtained from mucosal tissue of meat animals, mainly porcine intestine, and to a lesser extent bovine lung, its early stage production is poorly controlled, due to the source of the material (Figure 1). This problem came into sharp focus in 2008 when the presence of contaminating over-sulfated chondroitin sulfate in heparin, sourced from pigs, resulted in approximately 80 deaths in the USA [2]. This, coupled with the fact that only two doses are obtained per animal means that the demand for alternative and more controlled sources of heparin is high [3].

Figure 1. Process flow diagram for production of heparin and LMWH’s derived from porcine tissues/mucosa.

 

 

 

Figure 2. Biosynthetic pathway of heparin:The biosynthetic pathway includes the biosynthesis of polysaccharide backbone as well as the modification steps. The synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS polymerase. The backbone polysaccharide is then modified via five enzymatic modification steps (3)

 

Based on the related heparan sulfate (HS) biosynthetic pathway, Robert Linhardt’s and our groups have devised a chemoenzymatic process that begins with bacterial synthesis of the backbone structure. Escherichia coli K5 synthesizes a polysaccharide capsule consisting of repeating units of [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→)]n, known as heparosan [4,5,6]. Since heparosan is a precursor of heparin and heparan sulfate in eukaryotes, this provides an ideal starting point of the bioengineered heparin production process. The fermentation process involves the use of glucose as the sole carbon source and ammonium chloride as the sole nitrogen source. Initially developed and optimized on a 500 mL scale, the process was then scaled up to a 3 L fermentation and is currently at 100 L scale. Using fed-batch fermentation, with exponential feeding, a growth rate of 0.12 h-1 was achieved with a yield of 15 g/L heparosan [5].

Once extracted from the fermentation broth and purified the heparosan polysaccharide undergoes a series of chemoenzymatic modifications to make heparin. The first step is performed chemically and all other steps in the process use the enzymes listed in Figure 2, which have been cloned and overexpressed in E. coli. The heparosan polysaccharide generated through this process is analyzed by polyacrylamide gel electrophoresis (PAGE), 1D- and 2D-NMR and disaccharide analysis is done using LC-MS of heparinase-digested polysaccharide. At each point in the process the polysaccharide is analyzed by a combination of these methods [7,8,9]. Our recent study focused on endotoxin reduction in bioengineered heparin [10] and the optimization of the enzymatic reactions [11].

 

Reference

      1. F. Zhang, B. Yang, M. Ly, K. Solakyildirim, Z. Xiao, Z. Wang, J. M. Beaudet, A. Y. Torelli, J. S. Dordick, and R. J. Linhardt (2011) "Structural characterization of heparins from different commercial sources", Analytical and Bioanalytical Chemistry 401, 2793-2803.
      2. M. Guerrini, D. Beccati, Z. Shriver, A. M. Naggi, A. Bisio, I. Capila, J. Lansing, S. Guglieri, B. Fraser, A. Al-Hakim, S. Gunay, K. Viswanathan, Z. Zhang, L. Robinson, G. Venkataraman, L. Buhse, M. Nasr, J. Woodcock, R. Langer, R. Linhardt, B. Casu, G. Torri, and R. Sasisekharan (2008) "Oversulfated Chondroitin Sulfate is a major contaminant in Heparin associated with Adverse Clinical Events", Nature Biotechnology 26, 669-675.
      3. U Bhaskar, E Sterner, AM Hickey, A Onishi,F Zhang, JS Dordick, RJ Linhardt (2012) “Engineering of routes to heparin and related polysaccharides”, Applied Microbiology and Biotechnology 93, 1-16.
      4. Z.Wang, J.S.Dordick, and R.J.Linhardt (2011) "Escherichia coli K5 heparosan fermentation and improvement by genetic engineering", Bioengineered Bugs 2, 1-5.
      5. Z.Wang, M.Ly, F.Zhang, W. Zhong, A.Suen, A.M.Hickey, J.S.Dordick,and R.J.Linhardt (2010) "E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor", Biotechnology and Bioengineering 107, 964-973.
      6. M.Ly, Z.Wang, T.N.Laremore, F.Zhang, W.Zhong, D.Pu, D.V.Zagorevski, J.S.Dordick, and R.J.Linhardt (2011) "Analysis of E. coli K5 capsular polysaccharide heparosan", Analytical and Bioanalytical Chemistry 399, 737-745.
      7. P. Paul, J. Suwan, J. Liu, J. S. Dordick, and R. J. Linhardt (2012) "Recent advances in sulfotransferase enzyme activity assays", Analytical and Bioanalytical Chemistry 403, 1491-1500.
      8. Z. Zhang, S.A. McCallum, J. Xie, L. Nieto, F. Corzana, J.J.Barbero, M. Chen, J. Liu, and R.J.Linhardt (2008) “Solution Structures of Chemoenzymatically Synthesized Heparin and Its Precursors”, Journal of American Chemical Society 130, 12998–13007.
      9. Z. Wang, B. Yang, Z. Zhang, M. Ly, M. Takieddin, S. Mousa, J. Liu, J.S. Dordick, and R.J. Linhardt (2010) “Control of the heparosan N-deacetylation leads to an improved bioengineered heparin”, Applied Microbiology and Biotechnology 91, 91-99.
      10. Suwan J. Torelli A, Onishi A, Dordick JS, Lindhart RJ (2012) "Addressing endotoxin issues in bioengineered heparin", Biotechnol Appl Biochem 59, 420-428.
      11. U. Bhaskar, G. Li, L. Fu, A. Onishi, M. Suflita, J. S. Dordick, R. J. Lindhart (2014) "Combinatorial one-pot chemoenzymatic synthesis of heparin", Carbohydrate Polymers, in press.