An application of ultraviolet spectroscopy to study interactions in proteins solutions at high concentrations

作者：Thakkar Santosh V; Allegre Kevin M; Joshi Sangeeta B; Volkin David B; Middaugh C Russell^*

来源：Journal of Pharmaceutical Sciences, 2012, 101(9): 3051-3061.

DOI：10.1002/jps.23188

• protein
• high concentration
• interaction
• viscosity
• UV
• Vis spectroscopy
• light scattering

Studies of proteinprotein interactions (PPIs), especially in high-concentration solutions, have become increasingly important from a pharmaceutical perspective. Analytical methods used to study protein interactions, however, rely primarily on the detection of nonideality in relatively dilute (%26lt;50 mg/mL) solutions. We present here an application of variable-pathlength ultraviolet (UV)visible absorption spectroscopy to examine and better understand such interactions over a wide concentration range (5240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (?Abs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ?Abs, corrected for light scattering, was found to increase with protein concentration for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody). Because PPIs influence solution viscosity, we studied the correlation between ?Abs measurements and viscosity as a function of protein concentration. The magnitude of ?Abs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ?Abs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.

• 出版日期2012-9