Noninvasive determination of protein conformation in the solid state using near infrared (NIR) spectroscopy.
Fourier transform infrared (FTIR) spectroscopy is a powerful tool for monitoring structural changes in lyophilized protein formulations. However, direct measurement of IR spectra requires significant handling time and effort. The possibility of using near infrared (NIR) spectroscopy as a rapid and noninvasive alternative to FTIR is explored in this study. NIR and conventional FTIR spectra were collected for two model proteins, alpha-chymotrypsinogen A and cytochrome c, under conditions of varying stability and structural perturbation. NIR was then compared to FTIR and whereby calibration model was generated by partial least square (PLS) regression to correlate NIR data with FTIR spectra. There is a strong correlation of certain NIR bands with the amide I region of FTIR spectra. It appears that NIR can distinguish damage caused by elevated temperatures and freeze-drying stresses. The ability of sucrose to stabilize the structure of these two proteins can be detected by both methods. It appears that NIR spectroscopy has the potential to provide detailed information on the secondary structure of proteins in the solid state. However, many more examples will be needed to demonstrate fully the ability of NIR to replace FTIR as the standard tool for characterizing lyophilized protein formulations.
Bai
Shujun
S
Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Denver, Colorado, USA.
Nayar
Rajiv
R
Carpenter
John F
JF
Manning
Mark Cornell
MC
D002919:Chymotrypsinogen / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D005079:Excipients / Q000737:chemistry; D005612:Freeze Drying; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013395:Sucrose / Q000737:chemistry; D013696:Temperature
D005079:Excipients; D013395:Sucrose; D045304:Cytochromes c; D002919:Chymotrypsinogen