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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.

produce point-of-care diagnostic tools, which may be utilized for monitoring of biotechnological processes. Different conjugation techniques allowing a continuously adjustable surface coverage of laser-fabricated gold nanoparticles are presented. Maximum surface coverage was achieved using cationic peptides as aging-agents. The novel findings regarding optimized NBCs were transferred to two biotechnologically relevant analytes: ochratoxin A and ampicillin. This novel combination of ligand-free, laser-generated nanoparticles and aptamers could provide new strategies and applications for LFAs, while the basic materials themselves offer advantages with respect to their resource saving synthesis as well as the targeting of hazardous materials relevant in biotechnological processes using point-of-care-diagnostics.

Highlights An implemented high cell density cultivation process for biotechnological polysialic acid production is shown. Efficiency of polysialic acid production using E. coli K1 is significantly improved compared to state-of-the-art processes. Non-pathogenic E. coli BL21 is successfully engineered for production of α2,8- and α2,9-linked polySia. Differences of the α2,8- and α2,9-linked polysialic acid can be markedly visualized by the recorded NMR spectra.

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