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BIOMANUFACTURING


Bioengineered Heparin

Glycosaminoglycans (GAGs) are critical components of the stem cell niche and consist of long chain polymers of recurring disaccharide units usually composed of either D-glucosamine or D-galactosamine, and D-glucuronic acid or L-iduronic acid that when coupled to a core protein result in the formation of proteoglycans (PGs). Perhaps the most widely used of the GAG-based drugs is the highly sulfated anticoagulant heparin, which is used during surgery and dialysis, as a treatment of deep-vein thrombosis, and is used to coat indwelling catheters and other devices where the vascular system is exposed. Heparin is isolated from animal tissue, primarily pig intestines although it can be available from bovine and ovine intestines and other organs. Due to the animal source, the early stages of heparin production, e.g., generation of the raw heparin, occurs under poorly controlled, non-cGMP conditions, and purposeful contamination with related polysulfated polysaccharides was determined to result in the death of over 80 people in the U.S. in 2008. This has prompted the call for a non-animal sourced heparin.

In collaboration with Prof. Robert Linhardt, we have developed a biomanufacturing approach to a bioengineered heparin (Figure BH-1). Using the E. coli capsular polysaccharide heparosan, a series of chemical and enzymatic transformations are being optimized to generate a chemically and biologically equivalent (to USP) heparin. We have gained an improved understanding of the various sulfotransferases in the biosynthesis of heparin. Specifically, we developed a high-throughput microtiter-based colorimetric assay for elucidating the kinetic mechanism of heparin O-sulfotransferases (OSTs) acting on polysaccharide substrates using a given OST with arylsulfotransferase-IV and p-nitrophenylsulfate as sacrificial sulfate donor to regenerate the PAPS OST substrate [Paul et al. Anal. Bioanal. Chem. 403, 1491-1500 (2012)]. An example of the kinetic assessment is given in Figure BH-2 and results summarized in Table BH-1 [Sterner et al. Anal. Bioanal. Chem. 406, 525-536 (2013)].

  

Figure BH 1 Hparin Biomanufacturing

 

Figure BH-1. 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. References: Zhang et al. Anal. Bioanal. Chem. 401, 2793-2803 (2011); Vaidyanathan et al. Bioeng Transl Med. 2, 17-30 (2017); Bhaskar et al. Appl. Microbiol. Biotechnol. 93, 1-16 (2012); Wang et al. Bioeng. Bugs 2, 1-5 (2011); Wang et al. Biotechnol. Bioeng. 107, 964-973 (2010); Ly et al. Anal. Bioanal. Chem. 399, 737-745 (2011); Hickey et al. J. Biotechnol. 165, 175-177 (2013); Bhaskar et al. Carbohydr. Polym. 122, 399-407 (2014); Fu et al. J. Med. Chem. 60, 8673-8679 (2017).

 

Figure BH 2 Dual substrate kinetics

 

Figure BH-2. Dual-substrate kinetics of 6OST-3 on C5-epi treated N-sulfoheparosan. (A) Primary Lineweaver-Burk plot with respect to [PAPS]-1. (B) Primary Lineweaver-Burk plot with respect to [N-sulfoheparosan]-1. (C) Secondary replot of intercepts against [PAPS]-1. (D) Secondary replot of intercepts against [N-sulfoheparosan]-1 [Sterner et al. Anal. Bioanal. Chem. 406, 525-536 (2013].

 

Table BH-1. Detailed kinetic parameters of 6OST-3.

Figure53

More recent work has focused on the mechanistic understanding of glucuronosyl C5-epimerase (Glce), the first enzyme that acts on the polysaccharide substrate N-sulfoheparosan (NSH). Glce catalyzes the conversion of glucuronic acid to iduronic acid in NSH. Real-time 1H-NMR spectroscopy provides a highly sensitive method to quantify Glce kinetics (Figure BH-3).

BH 3rb

Figure BH-3. Anomeric proton of Glucuronic acid and Iduronic acid has different chemical shifts within the 1H-NMR spectrum and hence provides a sensitive method to detect Glce activity.

 

Collaborator:

Robert Linhardt – Rensselaer Polytechnic Institute