The 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.
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/5688 |
| Date | January 2010 |
| Creators | Bueche, Blaine |
| Contributors | Forbes, Robert T., Kuo, Mei-Chang |
| Publisher | University of Bradford, The School of Pharmacy |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| 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>. |
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