title: Characterization of protein higher order structure using vibrational circular dichroism spectroscopy.
abstract: Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a protein's HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling.
authors.affiliation: Bioanalytical Sciences, Biopharmaceutical Development R & D, GlaxoSmithKline, 709 Swedeland Rd, King of Prussia, PA 19406, USA.
authors.forename: Radhika P
authors.initials: RP
authors.lastname: Nagarkar
authors.forename: Brian M
authors.initials: BM
authors.lastname: Murphy
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Yu
authors.forename: Mark Cornell
authors.initials: MC
authors.lastname: Manning
authors.forename: Wasfi A
authors.initials: WA
authors.lastname: Al-Azzam
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012709:Serum Albumin / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D009211:Myoglobin; D012709:Serum Albumin; C527517:ofatumumab