Determination of the Structural Integrity and Stability of Polysialic Acid during Alkaline and Thermal Treatment

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.

Accelerated production of α2,8- and α2,9-linked polysialic acid in recombinant Escherichia coli using high cell density cultivation

“…The fluorescence signal in the chromatogram suggests that the actual maximum DP is still higher. These results are comparable to previous works [ 28 , 38 ]. The chromatograms of the recombinant strains show that the maximum chain length of the reference is comparable.…”

“…1 H spectra were recorded and compared with the spectrum of α2,8-polySia from E. coli K1. Comparison with the published chemical shifts of colominic acid (α2,8) and Nm C capsular polysaccharide (α2,9) allowed the assignment of all observed signals [ 11 , 28 , 44 ]. All 1 H-NMR ( Fig.…”

“…Since no targeted methods for endotoxin depletion were carried out in this process, the endotoxin load in the final product was also >100,000 EU/mg due to the higher cell densities after cultivation. The integration of alkaline incubation and subsequent anion exchange fractionation enables the production of highly pure polySia [ 28 , 36 ].…”

“…The glycosidic linkage patterns of the polySia produced were investigated by NMR spectroscopy. Sample preparation and measurement were performed as previously reported [ 28 ].…”

“…Contaminants such as host DNA and proteins (HCP) as well as endotoxins can be completely depleted by ethanol precipitation and clay minerals [ 27 ]. With a focus on a later medical application, these systems offer simple possibilities to produce polySia according to the quality requirements of "good manufacturing practice" (GMP) [ 28 ]. Nevertheless, the pathogenicity of E. coli K1 use makes it difficult to comply with and document the safety standards for patient protection according to GMP.…”