A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma.
Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-gamma (rhIFN-gamma) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-gamma aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-gamma and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-gamma native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-gamma against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.
Kendrick
B S
BS
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA. kendrick@amgen.com
Carpenter
J F
JF
Cleland
J L
JL
Randolph
T W
TW
D002850:Chromatography, Gel; D004926:Escherichia coli; D019791:Guanidine; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D011485:Protein Binding; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D013395:Sucrose; D013500:Surface Tension
D011994:Recombinant Proteins; D013395:Sucrose; D007371:Interferon-gamma; D019791:Guanidine