Mital, Malini; (2004) Polysialic acids: A tool for the optimisation of peptide and protein therapeutics. Doctoral thesis (Ph.D.), University College London.

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Abstract

Colominic acid (CA) is a low molecular weight derivative of polysialic acid (PSA), which is a naturally occurring biodegradable, highly hydrophilic polymer of N-acetylneuraminic acid. CA was proposed as an alternative to non-biodegradable mPEG for the delivery of proteins with short plasma half-lives, poor stability and high immunogenicity, thus facilitating their therapeutic potential. Here we add to the repertoire of proteins modified and introduce three 'new' smaller therapeutic peptides for polysialylation with a view to improving their in vitro stability, biological potency and in vivo pharmacokinetic properties. The biotherapeutics employed included, bovine polyclonal IgG and monoclonal IgG2a antibodies, which were chosen to model therapeutic Mabs which have the potential in treating cancers, autoimmune diseases and infections. The serine protease inhibitor, aprotinin chosen for the potential treatment of pancreatitis and haematological problems, insulin for treating diabetes and somatostatin (SS) for the treatment of growth disorders i.e. acromegaly. In addition, a novel strategy was developed in an attempt to further improve the efficiency and therapeutic value of the established method of polysialylation. Periodate oxidised CA was first coupled via Schiff base reductive amination to IgG (4.43%), aprotinin (29.20%), and insulin (63.33%) and yielded neoglycoproteins with a poor to moderate degree of modification based on the percentage of available amine residues modified with CA. Only SS obtained a quantitative yield of polysialylation. Introduction of the anionic detergent sodium dodecyl sulphate (SDS) into the coupling reaction seemed to enhance polysialylation and afforded bioconjugates with 1.5-3.0 fold increase in CA content. Size exclusion chromatography and electrophoresis were used to characterise and indicate the emergence of the neoglycoproteins. In vitro biological potency was mostly preserved for catalase (63-66%), IgG2a (82-94%) and aprotinin (59-87%) modified by either method of polysialylation. Furthermore, in vivo mean residence times of polysialylated and SDS-modified polysialylated IgG (45.46- 51.81h), aprotinin (22.23-24.73h) and insulin (22.81-23.26h) were significantly increased in comparison with their native counterparts. Polysialylation appears to confer stability to the biotherapeutics, maintain potency, delay plasma clearance, thereby improving their therapeutic potential in vivo.

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