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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.

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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

Histograms showing molar mass distributions for glucagon monomer and unfiltered aggregates analyzed by FFF/MALS. Initial glucagon solution evaluated immediately after dissolving in 0.01 N HCl is shown in a). Molar mass distribution after 22 hours incubation is shown in b). Molar mass of the large aggregate has grown by one order of magnitude over this period of time.
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Figure 9: Histograms showing molar mass distributions for glucagon monomer and unfiltered aggregates analyzed by FFF/MALS. Initial glucagon solution evaluated immediately after dissolving in 0.01 N HCl is shown in a). Molar mass distribution after 22 hours incubation is shown in b). Molar mass of the large aggregate has grown by one order of magnitude over this period of time.

Mentions: A high concentration unfiltered glucagon solution in 0.01 N HCl was again analyzed by FFF, both before and after 22 hours of incubation at 22°C. Initially, weight averaged molar mass of the monomer was analyzed by MALS to be 3650 g/mol, with a large aggregate peak of 1.2 × 105 g/mol. After the incubation period, the sample was observed to be partially gelled. When this sample was again analyzed by FFF/MALS, the molar mass of the large aggregate had increased significantly to 1.9 × 106 g/mol. The histograms in Figure 9 show the relative molar mass distributions for the two cases. The large aggregates are on the order of 1% of total injected mass, but the molar mass distributions for these peaks cannot be further analyzed due to extremely low UV signal strength.


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)

Histograms showing molar mass distributions for glucagon monomer and unfiltered aggregates analyzed by FFF/MALS. Initial glucagon solution evaluated immediately after dissolving in 0.01 N HCl is shown in a). Molar mass distribution after 22 hours incubation is shown in b). Molar mass of the large aggregate has grown by one order of magnitude over this period of time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Histograms showing molar mass distributions for glucagon monomer and unfiltered aggregates analyzed by FFF/MALS. Initial glucagon solution evaluated immediately after dissolving in 0.01 N HCl is shown in a). Molar mass distribution after 22 hours incubation is shown in b). Molar mass of the large aggregate has grown by one order of magnitude over this period of time.
Mentions: A high concentration unfiltered glucagon solution in 0.01 N HCl was again analyzed by FFF, both before and after 22 hours of incubation at 22°C. Initially, weight averaged molar mass of the monomer was analyzed by MALS to be 3650 g/mol, with a large aggregate peak of 1.2 × 105 g/mol. After the incubation period, the sample was observed to be partially gelled. When this sample was again analyzed by FFF/MALS, the molar mass of the large aggregate had increased significantly to 1.9 × 106 g/mol. The histograms in Figure 9 show the relative molar mass distributions for the two cases. The large aggregates are on the order of 1% of total injected mass, but the molar mass distributions for these peaks cannot be further analyzed due to extremely low UV signal strength.

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