Abstract
Polysialic acid (polySia) is a linear homopolymer of varying chain lengths that exists mostly on the outer cell membrane surface of certain bacteria, such as Escherichia coli (E. coli) K1. PolySia, with an average degree of polymerization of 20 (polySia avDP20), possesses material properties that can be used for therapeutic applications to treat inflammatory neurodegenerative diseases. The fermentation of E. coli K1 enables the large-scale production of endogenous long-chain polySia (DP ≈ 130) (LC polySia), from which polySia avDP20 can be manufactured using thermal hydrolysis. To ensure adequate biopharmaceutical quality of the product, the removal of byproducts and contaminants, such as endotoxins, is essential. Recent studies have revealed that the long-term incubation in alkaline sodium hydroxide (NaOH) solutions reduces the endotoxin content down to 3 EU (endotoxin units) per mg, which is in the range of pharmaceutical applications. In this study, we analyzed interferences in the intramolecular structure of polySia caused by harsh NaOH treatment or thermal hydrolysis. Nuclear magnetic resonance (NMR) spectroscopy revealed that neither the incubation in an alkaline solution nor the thermal hydrolysis induced any chemical modification. In addition, HPLC analysis with a preceding 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization demonstrated that the alkaline treatment did not induce any hydrolytic effects to reduce the maximum polymer length and that the controlled thermal hydrolysis reduced the maximum chain length effectively, while cost-effective incubation in alkaline solutions had no adverse effects on LC polySia. Therefore, both methods guarantee the production of high-purity, low-molecular-weight polySia without alterations in the structure, which is a prerequisite for the submission of a marketing authorization application as a medicinal product. However, a specific synthesis of low-molecular-weight polySia with defined chain lengths is only possible to a limited extent.
Highlights
Polysialic acid is a linear homopolymer consisting of sialic acid monomers
Both procedures represent simple and efficient ways to remove the endotoxin burden of produced Polysialic acid (polySia) to achieve a product of pharmaceutical grade
Via incubation in alkaline solutions and recovery using anion exchange (AEX) membrane adsorbers, the endotoxin burden in the final product was cheaply and reduced to levels down to 2 EU per mg, which is in the range for medical applications
Summary
Polysialic acid (polySia) is a linear homopolymer consisting of sialic acid monomers. Sialic acid is a derivative of neuraminic acid, which normally contains an acetylated amino function on the C5 atom, leading to N-acetylneuraminic acid (Neu5Ac) [1,2]. Some prokaryotes like Neisseria meningitis (N. meningitis) or Escherichia (E. coli) K1 synthesize a human-identical α-(2,8)-linked form of meningitis or Escherichia (E. coli) K1and synthesize a human-identical polySia (Figure 1A) Α-(2,8)-linked form of polySia (Figure 1A) as a capsular polysaccharide and virulence factor. Structure the structure of α-(2,8)-linked derivates polysialic acid:. Homopolymer of α-(2,8)-linked neuraminic acid, and (C) the lactonized structure of α-(2,8)-linked polySia Some prokaryotes like Neisseria meningitis (N. meningitis) or Escherichia (E. coli) K1 synthesize a human-identical α-(2,8)-linked form of meningitis or Escherichia (E. coli) K1and synthesize a human-identical polySia (Figure 1A) (N. as meningitis)
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