Insulin Amyloid Formation: Effects of additives and environmental factors encountered during production scale HPLC purification
Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
Insulin is a peptide hormone that regulates blood sugar levels and is used to treat diabetes. In acidic buffers, high temperature, and at high concentration, insulin aggregates to form insoluble amyloid fibrils. This is problematic in industrial production of insulin, where aggregation during HPLC purification results in clogged columns and reduced separation capacity. Insulin amyloid formation has mainly been studied at low pH (~2); the influence of protein concentration, organic modifiers, and excess surfaces and particles at intermediate (~4) or neutral pH is less clear, albeit highly relevant to understand insulin fibrillation in industrial production settings. To address this, Thioflavin-T (ThT) fluorescence was used to study insulin fibrillation under different experimental conditions in vitro.Moreover, insulin solubility and thermodynamic stability was determined across a range of relevant solution pH using spectroscopic techniques, including circular dichroism. The main conclusions of this work are that insulin aggregation is faster at low and neutral pH, whereas aggregation is slower in the intermediate range between pH 4 and 6. Further, increasing amounts of ethanol or isopropanol has a retarding effect on fibril formation at concentrations up to 30% (v/v). Presence of hydrophobic silica particles enhance insulin fibrillation, which explains the observations of this adverse reaction during HPLC purifications. Also, this work concludes that the stability of insulins secondary structure is unaffected by pH.
This project has given insight into how insulin aggregation could be avoided and has thereby enabled further work for new ideas on how clogged columns could be restored by dissolving insulin aggregates.
Place, publisher, year, edition, pages
UPTEC K, ISSN 1650-8297 ; 16006
Natural Sciences Analytical Chemistry
IdentifiersURN: urn:nbn:se:uu:diva-298411OAI: oai:DiVA.org:uu-298411DiVA: diva2:945915
AkzoNobel & Chalmers University of Technology
Master Programme in Chemical Engineering
2016-06-01, Uppsala, 13:15 (English)
Esbjörner Winters, Elin, Assistant Professor
Petterson, CurtWesterlund, Douglas