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.

While a number of PEGylated proteins have been studied for injectable applications and reviewers have used this data to speculate possible oral delivery improvements, a detailed investigation of PEGylated insulin for oral delivery and the development of an optimized pH-sensitive carrier for PEGylated insulin conjugates had yet to be accomplished. In order to proceed with oral delivery study, improvements in yield, with respect to previous PEGylation methods were necessary to enable the completion of high throughput drug delivery studies. Subsequently, a reaction scheme for the covalent attachment of PEG to insulin using nitrophenyl carbonate-PEG was developed. It was demonstrated that this reaction occurred at a 1:1 ratio and was site specific at the B29Lys position. A P(MAA-g-EG) hydrogel carrier was developed to optimize loading and release behavior for PEGylated insulin. It was demonstrated that the density and length of polymer grafts affected both loading and release behavior of PEGylated insulin. The best performing grafted polymers had a 3:1 methacrylic acid: ethylene glycol (MAA:EG) ratio and achieved loading efficiencies from 96% to nearly 100%. With respect to release, polymer particles containing fewer, but longer grafts shown to release faster than polymers with shorter grafts with the same MAA:EG ratio. Finally, the effects of PEGylation on intestinal absorption was investigated using an intestinal epithelial model as well as a rat model. It was demonstrated that PEGylated insulin in the presence of P(MAA-g-EG) microparticles did not significantly alter the tight junctions over unmodified insulin. However, the conjugate permeabilities across the membrane were reduced. The pharmacological availability (PA) was then verified by injecting the insulin conjugates subcutaneously in fasted Sprague-Dawley rats. It was determined that PEG 1000 insulin (1KPI) had a PA roughly equivalent to insulin, while it was reduced by 59% for 2KPI and by 81% for 5KPI. The effectiveness of utilizing PEGylated insulin as an oral drug delivery candidate was evaluated with a closed loop intestinal study, in which PEGylated insulin or insulin in solution was delivered directly to the jejunum. It was shown that 1KPI and insulin performed identically; with a pharmacological availability of 0.56%. 2KPI, however improved the pharmacological availability of insulin by 2.8 times. These results demonstrate that PEGylation holds promise for improving the oral delivery of proteins. / text

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.