title: Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.
abstract: We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.
authors: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors: Sampathkumar
authors: S
authors: Krishnan
authors: Eva Y
authors: EY
authors: Chi
authors: Jonathan N
authors: JN
authors: Webb
authors: Byeong S
authors: BS
authors: Chang
authors: Daxian
authors: D
authors: Shan
authors: Merrill
authors: M
authors: Goldenberg
authors: Mark C
authors: MC
authors: Manning
authors: Theodore W
authors: TW
authors: Randolph
authors: John F
authors: JF
authors: Carpenter
mesh_terms: D011232:Chemical Precipitation; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry; D019281:Dimerization; D016179:Granulocyte Colony-Stimulating Factor / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012996:Solutions; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose / Q000737:chemistry; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D012996:Solutions; D016179:Granulocyte Colony-Stimulating Factor; D013395:Sucrose; D006859:Hydrogen; D003903:Deuterium