Abstract
BackgroundGlucagon 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.ResultsHigh 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.ConclusionThe 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.
Highlights
Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia
In order to demonstrate the ability of fieldflow fractionation (FFF)/multiangle light scattering (MALS) to speciate aggregates, and to provide absolute molecular weight determination, a mixture of five different protein standards was analyzed: bovine serum albumin (BSA), alcohol dehydrogenase, β-amylase, apoferritin, and thyroglobulin
The results of these FFF/MALS experiments using protein standards demonstrate that FFF is an effective method for separating proteins and protein aggregates
Summary
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. 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. Glucagon has several uses as a therapeutic agent, including the emergency treatment of hypoglycemia [1]. The medium in which lyophilized glucagon is normally solubilized, this gel formation has been shown to result from the growth of fibrillar aggregates [2]. Understanding the aggregation process is important (page number not for citation purposes)
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