Show simple item record

dc.contributor.advisorForbes, Robert T.
dc.contributor.advisorKuo, Mei-Chang
dc.contributor.authorBueche, Blaine*
dc.date.accessioned2013-11-20T17:58:39Z
dc.date.available2013-11-20T17:58:39Z
dc.date.issued2013-11-20
dc.identifier.urihttp://hdl.handle.net/10454/5688
dc.description.abstractThe objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin¿s systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin¿s charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID¿s in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.en_US
dc.language.isoenen_US
dc.rights<a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>.eng
dc.subjectSpray dryingen_US
dc.subjectInsulinen_US
dc.subjectPegylated insulinen_US
dc.subjectIon exchangeen_US
dc.subjectChromatographyen_US
dc.subjectHexameren_US
dc.subjectZinc-free insulinen_US
dc.subjectLight scatteringen_US
dc.subjectMALSen_US
dc.titleThe Effect of PEG-Insulin and Insulin Hexamer Assembly on Stability in Solution and Dry Powders. Hexamer Assembly of PEGylated-Insulin and Insulin Studied by Multi-Angle Light Scattering to Rationally Choose the pH and Zinc Content for Analytical Methods and Formulations of Dry Powders.en_US
dc.type.qualificationleveldoctoralen_US
dc.publisher.institutionUniversity of Bradfordeng
dc.publisher.departmentThe School of Pharmacyen_US
dc.typeThesiseng
dc.type.qualificationnamePhDen_US
dc.date.awarded2010
refterms.dateFOA2018-07-19T12:10:16Z


Item file(s)

Thumbnail
Name:
B. Bueche Thesis.pdf
Size:
7.340Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record