Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.
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.
Krishnan
Sampathkumar
S
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Chi
Eva Y
EY
Webb
Jonathan N
JN
Chang
Byeong S
BS
Shan
Daxian
D
Goldenberg
Merrill
M
Manning
Mark C
MC
Randolph
Theodore W
TW
Carpenter
John F
JF
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
D011994:Recombinant Proteins; D012996:Solutions; D016179:Granulocyte Colony-Stimulating Factor; D013395:Sucrose; D006859:Hydrogen; D003903:Deuterium