Addressing endotoxin issues in bioengineered heparin

Suwan, Jiraporn; Torelli, Amanda Y.; Ōnishi, Akira; Dordick, Jonathan S.; Linhardt, Robert J.

doi:10.1002/bab.1042

Cited by 6 publications

(3 citation statements)

References 32 publications

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“…A significant reduction in endotoxin levels (2.5 log) was recorded for chitosan‐purified heparosan while SAX purified heparosan still contained a high endotoxin load. This endotoxin reduction is important, as use of chitosan in the bioengineered heparin process can circumvent the need for a change in enzyme expression systems to mitigate endotoxin risk arising due to use of E. coli based expression systems . Further characterization of this process, however, is necessary to ascertain selectivity.…”

Section: Resultsmentioning

confidence: 99%

“…This endotoxin reduction is important, as use of chitosan in the bioengineered heparin process can circumvent the need for a change in enzyme expression systems to mitigate endotoxin risk arising due to use of E. coli based expression systems. [49][50][51][52] Further characterization of this process, however, is necessary to ascertain selectivity. The purified products, appearing as a single peak, suggest a higher molecular weight of heparosan compared to ammonium sulfate purified heparosan derived from the same fermentation broth.…”

Section: Characterization Of Heparosan Products Purified Using Sax Chmentioning

confidence: 99%

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A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

Bhaskar

Hickey

et al. 2015

Biotechnol Progress

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The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical Ndeacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatographymass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition.

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Section: Resultsmentioning

confidence: 99%

Section: Characterization Of Heparosan Products Purified Using Sax Chmentioning

confidence: 99%

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

Bhaskar

Hickey

et al. 2015

Biotechnol Progress

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“…DEAE-Sephadex chromatography is a method reported to efficiently remove LPS from proteins (Hou et al 1990). Suwan et al (Suwan et al 2012) have reported that anion-exchange chromatography was efficient to remove endotoxins from heparosan (a non-sulfated precursor of heparin), but not from sulfated heparin. The inefficiency was attributed to the binding of heparin to DEAE due to the low pKa value of the sulfate groups.…”

Section: Discussionmentioning

confidence: 99%

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them

Zykwinska

Sinquin

et al. 2017

Appl Microbiol Biotechnol

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Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content.

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Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N‐acetylglucosamine supplementation and kinetic modeling

Nehru,

Balakrishnan,

Swaminathan

et al. 2024

Biotech and App Biochem

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Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin—an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30 g L−1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N‐acetylglucosamine (S2) resulted in a significant 1.6‐fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi‐substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.

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Addressing endotoxin issues in bioengineered heparin

Cited by 6 publications

References 32 publications

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them

Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N‐acetylglucosamine supplementation and kinetic modeling

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