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

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.

Amyloid structure – one but not the same: the many levels of fibrillar polymorphism

“…Several studies have been aimed at characterizing prefibrillar intermediate species of glucagon and oligomeric structures have been reported in AFM studies [61,62]. However, according to data from field flow fractionation [79], NMR [80], SAXS [63] and dynamic light scattering [44], the benign α‐helical trimer, which is in rapid equilibrium with monomers [44,64,81] and crystallizes readily [14,82], is the only oligomeric structure that forms at detectable levels at pH 2.5. It is possible that a shift to pH 5.5, where the molecules have an average charge of +1, could allow glucagon to form more stable toxic oligomeric species.…”

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

“…The physical instability of glucagon causes problems in formulation, and in the delivery of its pharmaceutical product. Hoppe et al [67] investigated the initial aggregation formation, i.e. the seed nuclei of glucagon in acidic and alkaline media by AsFlFFF-MALS, SLS and DLS.…”

“…Since then, separation speed, and separation efficiencies have been greatly improved. The use of MALS detector with AsFlFFF/FlFFF provides accurate size, shape and molar mass information of biopolymers including proteins and protein aggregates, without the need of calibration with standards of known molar masses [66,67].…”

Razdvajanje protočnim poljem u analizi kompleksnih bioloških uzoraka

“…Viskozni i slabo topljivi proteinski agregati predstavljaju problem kod gel-filtracijske tehnike zbog začepljivanja pora u koloni, dok kod tehnike FFF može doći do začepljenja pora na akumulacijskoj stijenci, a obje tehnike se često primjenjuju za razdvajanje agregata, oligomera i monomera proteina. 29 Tehnika FlFFF je najčešće primjenjivana tehnika pri analizi proteina i proteinskih kompleksa jer se difuzijski koeficijent može povezati s veličinom i oblikom proteina. Na taj je način moguće pratiti vrlo male promjene u strukturi proteina i proteinskih kompleksa.…”