The stability of bFGF against thermal denaturation.
The influence of sulphated ligand and pH on thermal denaturation of basic fibroblast growth factor (bFGF) was investigated by differential scanning calorimetry (DSC), and verified by fluorescence spectrophotometry. Purity of bFGF before and after heat denaturation was assessed by SDS-PAGE analysis. In DSC studies the samples were heated to 95 degrees C. The midpoint of the temperature change in the thermogram was designated as Tm. Sulphated ligand experiments were undertaken in potassium phosphate (pH 6.5) and sodium acetate buffers. Control thermograms (with no ligand) showed a Tm at 59 degrees C in potassium phosphate buffer. Higher Tm values were noted as sulphated ligand concentration was increased. Similarly when heparin was added, the Tm moved to a higher temperature. A ratio as low as 0.3:1 of heparin to bFGF, increased the Tm to 90 degrees C, which is a 31 degrees C shift in Tm. The effect of pH on thermal denaturation of bFGF was studied in a citrate-phosphate-borate buffer system. A shift in Tm from 46 to 65 degrees C was observed as the pH is changed from 4 to 8. Changes in protein conformation as a function of pH were monitored by fluorescence spectroscopy. It was found that a pH range from 5 to 9 is optimal for the stability of bFGF formulations. In a stability study it was noted that heparin protected bFGF from thermal denaturation only at high temperature.
Vemuri
S
S
Scios Nova Inc., Mountain View, CA 94043.
Beylin
I
I
Sluzky
V
V
Stratton
P
P
Eberlein
G
G
Wang
Y J
YJ
D002021:Buffers; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives / Q000737:chemistry; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013395:Sucrose / Q000031:analogs & derivatives / Q000737:chemistry; D013678:Technology, Pharmaceutical
D002021:Buffers; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C013063:inositol hexasulfate; D007294:Inositol; D013395:Sucrose; D006493:Heparin; C053081:sucrose octasulfate