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

Hoppe CC, Nguyen LT, Kirsch LE, Wiencek JM - J Biol Eng (2008)

Bottom Line: In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, University of South Florida, Tampa, FL, 33520, USA. jwiencek@eng.usf.edu.

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.

No MeSH data available.


Related in: MedlinePlus

Raw scattered light intensity from ALV SLS detector vs. time for filtered and unfiltered glucagon preparations in dilute HCl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483260&req=5

Figure 7: Raw scattered light intensity from ALV SLS detector vs. time for filtered and unfiltered glucagon preparations in dilute HCl.

Mentions: The effect of pre-existing aggregates on aggregation rate was further investigated by measuring the raw light scattering signal intensity of freshly prepared 4 mg/mL glucagon solutions in 0.01 N HCl over a period of three days (Fig. 7). The rapid increase in light scattering intensity for the unfiltered glucagon preparation suggests that the presence of initial aggregates serve to nucleate the aggregation process. Conversely, the filtered acid glucagon preparation with the absence of initial aggregates stayed stable in solution for 50 hours.


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

Hoppe CC, Nguyen LT, Kirsch LE, Wiencek JM - J Biol Eng (2008)

Raw scattered light intensity from ALV SLS detector vs. time for filtered and unfiltered glucagon preparations in dilute HCl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483260&req=5

Figure 7: Raw scattered light intensity from ALV SLS detector vs. time for filtered and unfiltered glucagon preparations in dilute HCl.
Mentions: The effect of pre-existing aggregates on aggregation rate was further investigated by measuring the raw light scattering signal intensity of freshly prepared 4 mg/mL glucagon solutions in 0.01 N HCl over a period of three days (Fig. 7). The rapid increase in light scattering intensity for the unfiltered glucagon preparation suggests that the presence of initial aggregates serve to nucleate the aggregation process. Conversely, the filtered acid glucagon preparation with the absence of initial aggregates stayed stable in solution for 50 hours.

Bottom Line: In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, University of South Florida, Tampa, FL, 33520, USA. jwiencek@eng.usf.edu.

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.

No MeSH data available.


Related in: MedlinePlus