title: An application of ultraviolet spectroscopy to study interactions in proteins solutions at high concentrations.
abstract: Studies of protein-protein 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 (<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 (5-240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (ΔAbs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ΔAbs, 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 ΔAbs measurements and viscosity as a function of protein concentration. The magnitude of ΔAbs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ΔAbs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Santosh V
authors.initials: SV
authors.lastname: Thakkar
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Allegre
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D000042:Absorption; D000911:Antibodies, Monoclonal / Q000737:chemistry; D008027:Light; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013678:Technology, Pharmaceutical / Q000379:methods*; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal; D011506:Proteins; D012710:Serum Albumin, Bovine; D009113:Muramidase