Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: a bGCSF case study.

In protein and macromolecule pharmaceutical formulation development, the amount of information initially gathered about a drug's physical and chemical properties under different conditions is often quite limited. This generally requires more intensive studies using a variety of techniques if problems arise later in the development process. We propose a supplementary approach involving a comprehensive examination of a protein by derivative absorbance spectroscopy in conjunction with other methods and the subsequent construction of a phase diagram that permits the determination of optimal formulation conditions. Using bovine granulocyte colony stimulating factor (bGCSF) as a model protein, a thorough characterization is performed using high-resolution second-derivative absorbance spectroscopy. Derivative ultraviolet absorbance data are used to construct an empirical phase diagram for bGCSF using a multidimensional phase space approach. Between pH 2 and 7, and from 10 degrees to 90 degrees C, bGCSF is found to adopt more than six distinct structural phases. Surprisingly, the combination of the phase diagram approach with derivative absorbance data identifies phase boundaries that are not apparent upon initial examination of complementary biophysical data (previous article in this issue). The simplicity and pharmaceutical utility of this approach are discussed. Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association