Spectroscopic analysis of highly concentrated suspensions of bovine somatotropin in sesame oil.
Spectroscopy was employed to analyze the structural and thermal stability of highly concentrated oil suspensions of bovine somatotropin (bST). These methods were then compared with more dilute aqueous solutions (1 and 10 mg/mL). All oil suspensions were opaque, viscous, and highly concentrated in bST (>300 mg/mL) and thus provided unique analytical challenges. Using front surface fluorescence and ATR-FTIR spectroscopy, protein structure and stability could be directly monitored in this environment. Differences were detected in structure between concentrated oil and dilute aqueous formulations. Fluorescence spectroscopy found that bST was highly thermally stabile within oil suspensions, since minimal changes in emission peak maxima and emission intensity were observed with increasing temperature when compared to dilute solutions. It was also observed that the amount of aggregate in a sample had some effect on the fluorescence spectra. As the amount of aggregated protein increased, the emission peak maximum and emission intensity changed. Employing ATR-FTIR, the secondary structure was examined with increasing temperature. The secondary structure of bST was also found to be very thermally stabile since no change in relative amount of helix/random structure is observed up to 70 degrees C while significant losses are observed in aqueous solution. This study demonstrates that conformational stability can be directly analyzed within highly concentrated, opaque environments using slight modifications of conventional methods.
Harn
Nicholas R
NR
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas, Lawrence, Kansas 66047, USA.
Jeng
Yunhua N
YN
Kostelc
James G
JG
Middaugh
C Russell
CR
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004355:Drug Stability; D013006:Growth Hormone / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012715:Sesame Oil / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013535:Suspensions; D013696:Temperature; D014783:Viscosity; D014867:Water / Q000737:chemistry
D013535:Suspensions; D014867:Water; D012715:Sesame Oil; D013006:Growth Hormone