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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

Relative intensity of Rayleigh ratio from MALS detector for filtered and unfiltered glucagon solutions in 0.01 N HCl. Monomer and aggregates have been separated by asymmetric flow FFF in the unfiltered sample. Aggregates have been removed from the solution in the filtered sample.
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Figure 8: Relative intensity of Rayleigh ratio from MALS detector for filtered and unfiltered glucagon solutions in 0.01 N HCl. Monomer and aggregates have been separated by asymmetric flow FFF in the unfiltered sample. Aggregates have been removed from the solution in the filtered sample.

Mentions: FFF/MALS experiments were performed to verify the presence of large initial aggregates upon initial mixing of glucagon in 0.01 N HCl, and to confirm that the large aggregates can be removed by filtration, as previously indicated by DLS results. The 100 nm aggregate population was not concentrated enough to be detectable at the low concentrations and small injection volumes typically used with chromatographic methods. Therefore 250 μL of a 14 mg/ml glucagon solution was injected into the FFF channel for these experiments both before and after filtration with a 0.1 μm filter. Elution of a large aggregate peak can be observed in the unfiltered sample, but not in the filtered sample (Fig. 8). The use of a UV detector downstream of the FFF unit allowed for the verification that the large particles are indeed protein, since this peak exhibits measurable UV absorbance at 278 nm.


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)

Relative intensity of Rayleigh ratio from MALS detector for filtered and unfiltered glucagon solutions in 0.01 N HCl. Monomer and aggregates have been separated by asymmetric flow FFF in the unfiltered sample. Aggregates have been removed from the solution in the filtered sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Relative intensity of Rayleigh ratio from MALS detector for filtered and unfiltered glucagon solutions in 0.01 N HCl. Monomer and aggregates have been separated by asymmetric flow FFF in the unfiltered sample. Aggregates have been removed from the solution in the filtered sample.
Mentions: FFF/MALS experiments were performed to verify the presence of large initial aggregates upon initial mixing of glucagon in 0.01 N HCl, and to confirm that the large aggregates can be removed by filtration, as previously indicated by DLS results. The 100 nm aggregate population was not concentrated enough to be detectable at the low concentrations and small injection volumes typically used with chromatographic methods. Therefore 250 μL of a 14 mg/ml glucagon solution was injected into the FFF channel for these experiments both before and after filtration with a 0.1 μm filter. Elution of a large aggregate peak can be observed in the unfiltered sample, but not in the filtered sample (Fig. 8). The use of a UV detector downstream of the FFF unit allowed for the verification that the large particles are indeed protein, since this peak exhibits measurable UV absorbance at 278 nm.

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