Irreversible aggregation of recombinant bovine granulocyte-colony stimulating factor (bG-CSF) and implications for predicting protein shelf life.
The kinetics of irreversible aggregation of bovine Granulocyte-Colony Stimulating Factor (bG-CSF) in solution were investigated as a function of temperature (T), concentration, and pH, and analyzed in terms of an Extended Lumry-Eyring model of protein aggregation proceeding via a non-native conformational state. In the spirit of classic Lumry-Eyring models, the observed kinetics are separated into contributions from thermodynamic or conformational stability of unaggregated native and non-native states, and the intrinsic aggregation kinetics of non-native molecules. It is found that a detailed treatment of the intrinsic kinetics coupled with a two-state approximation of the reversible unfolding transition is sufficient to allow quantitative prediction of low-T stability from high-T data despite highly non-Arrhenius kinetics. Accounting for shifts in conformational equilibrium quantitatively captures the non-Arrhenius T dependence, without requiring the assumption of a change in the rate-determining step with T. From a more general perspective, the observed aggregation behavior of bG-CSF is consistent with the rate-determining step being aggregation at T below a crossover temperature T(x) that is inversely related to initial protein concentration. Above T(x), irreversible unfolding is presumably the rate-determining step. The results illustrate that protein aggregation kinetics can, in principle, be predicted quantitatively from so-called accelerated data provided the thermodynamic and kinetic components can be separately extrapolated to longer term storage conditions.
Roberts
Christopher J
CJ
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA. roberts@che.udel.edu
Darrington
Richard T
RT
Whitley
Maureen B
MB
D000465:Algorithms; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004355:Drug Stability; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics
D016179:Granulocyte Colony-Stimulating Factor