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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

Debye plot of glucagon immediately upon dissolving in 0.01 N NaOH. Measurements obtained by static light scattering at 30°C and a 90° scattering angle. Linear regression model generated using Kc/R90 and glucagon concentration yielded a y-intercept of 2.6 (± 0.2) × 10-4, which corresponds to a Mw = 3831 (± 275).
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Figure 5: Debye plot of glucagon immediately upon dissolving in 0.01 N NaOH. Measurements obtained by static light scattering at 30°C and a 90° scattering angle. Linear regression model generated using Kc/R90 and glucagon concentration yielded a y-intercept of 2.6 (± 0.2) × 10-4, which corresponds to a Mw = 3831 (± 275).

Mentions: Filtered glucagon solutions were analyzed immediately after mixing by SLS to determine the weight-averaged molar mass (Mw) using the Debye equation (Eqn. 1). The excess Rayleigh ratio at 90° scattering angle, R90, was measured over a range of known concentrations. By plotting values of Kc/R90 over a range of concentrations, the Mw was determined from the inverse of the y-intercept value. Debye plots of Kc/R90 as a function of concentration for acidic and alkaline glucagon solutions are shown in Figure 4 and Figure 5. The Mw of soluble glucagon in the acidic pH region was determined to be 3539 ± 372 g/mol. Glucagon in alkaline solutions exhibited a slightly higher Mw, 3831 ± 275 g/mol. This slightly higher alkaline Mw may indicate the presence of higher molecular weight aggregates in the alkaline solutions. In order to verify this, DLS was utilized to further probe these samples.


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)

Debye plot of glucagon immediately upon dissolving in 0.01 N NaOH. Measurements obtained by static light scattering at 30°C and a 90° scattering angle. Linear regression model generated using Kc/R90 and glucagon concentration yielded a y-intercept of 2.6 (± 0.2) × 10-4, which corresponds to a Mw = 3831 (± 275).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Debye plot of glucagon immediately upon dissolving in 0.01 N NaOH. Measurements obtained by static light scattering at 30°C and a 90° scattering angle. Linear regression model generated using Kc/R90 and glucagon concentration yielded a y-intercept of 2.6 (± 0.2) × 10-4, which corresponds to a Mw = 3831 (± 275).
Mentions: Filtered glucagon solutions were analyzed immediately after mixing by SLS to determine the weight-averaged molar mass (Mw) using the Debye equation (Eqn. 1). The excess Rayleigh ratio at 90° scattering angle, R90, was measured over a range of known concentrations. By plotting values of Kc/R90 over a range of concentrations, the Mw was determined from the inverse of the y-intercept value. Debye plots of Kc/R90 as a function of concentration for acidic and alkaline glucagon solutions are shown in Figure 4 and Figure 5. The Mw of soluble glucagon in the acidic pH region was determined to be 3539 ± 372 g/mol. Glucagon in alkaline solutions exhibited a slightly higher Mw, 3831 ± 275 g/mol. This slightly higher alkaline Mw may indicate the presence of higher molecular weight aggregates in the alkaline solutions. In order to verify this, DLS was utilized to further probe these samples.

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