Research

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

Cindy C Hoppe¹ , Lida T Nguyen² , Lee E Kirsch² and John M Wiencek³

¹Department of Chemical & Biochemical Engineering, University of Iowa, Iowa City, IA, 52242, USA

²Division of Pharmaceutics, University of Iowa, Iowa City, IA, 52242, USA

³Department of Chemical Engineering, University of South Florida, Tampa, FL, 33520, USA

author email corresponding author email

Journal of Biological Engineering 2008, 2:10doi:10.1186/1754-1611-2-10

Published:	9 July 2008

Abstract

Background

Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.

Results

High molecular weight aggregates of glucagon were detected in otherwise monomeric solutions using light scattering techniques. These aggregates were detected upon initial mixing of glucagon powder in dilute HCl and NaOH. In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process. Field-flow fractionation was used to separate aggregates from monomeric glucagon and determine relative mass. The molar mass of the large aggregates was shown to grow appreciably over time as the glucagon solutions gelled.

Conclusion

The results of this study indicate that initial glucagon solutions are predominantly monomeric, but contain small quantities of large aggregates. These results suggest that the initial aggregates are seed nuclei, or intermediates which catalyze the aggregation process, even at low concentrations.