An empirical phase diagram approach to the characterization of therapeutic proteins

Rational formulation development for protein-based therapeutics requires an understanding of the critical properties of the target molecule in significant experimental depth. Comprehensive pre-formulation studies are necessary to achieve this goal, in which a wide variety of analytical techniques are employed to test the drug's susceptibility to various pharmaceutically relevant stresses. One potential problem with such a comprehensive analysis is that the large amount of data generated can be difficult to interpret due to the diversity of the experiments conducted.;An empirical phase diagram (EPD) approach has been developed as a novel data analysis method to address this problem. A multi-dimensional phase space approach is used to find patterns among large data sets. The results are plotted in an RGB color scheme to allow visualization and interpretation of the data. Empirical phases can then be identified as regions of homogeneous color with substantially uniform and coherent structural data. Actual physical states of the protein can be assigned to each of the empirical phases based on the individual raw data.;Five pharmaceutical proteins with varying size and structure were extensively characterized to test the overall utility of the EPD approach. For the first time, phase diagrams were constructed using heterogeneous data sets, including data from a variety of biophysical measurement such as derivative UV absorbance, far-UV circular dichroism, intrinsic and ANS fluorescence spectroscopy. Combining multiple techniques can provide a more global picture of changes to the protein structure. Phase diagrams were also created for proteins in the presence of different types of stabilizers, illustrating their differential effects on the target protein.;A generic method based on the EPD approach can be established to identify stabilizers for protein therapeutics. The empirical phase diagram can provide an intuitive description of the structural stability of proteins over a wide pH and temperature range. Appropriate pH and temperature conditions can then be determined for subsequent high throughput excipients screening processes based on the phase diagram. This process provides information on which to base recommendations for initial formulations for further development.;In future studies, experimental data reflecting chemical stability, dynamic structural properties and the biological activity of proteins will be incorporated to construct more diverse phase diagrams, which can provide more comprehensive information to assist in the rational development of protein formulations.