Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA and macromolecular complexes such as viruses, virus-like particles and adjuvanted antigens is often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods, since each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecule's physical state as a function of temperature, solvent conditions, and other stress variables. We present a tutorial on the mathematical methodology, an overview of some of the experimental methods typically used, and examples of some of the previous major formulation applications. We also explore novel applications of EPDs including potential new mathematical approaches as well as possible new biopharmaceutical applications such as analytical comparability, chemical stability, and protein dynamics.

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The field of pharmaceutical chemistry is currently struggling with the question of how to relate changes in the physical form of a macromolecular biopharmaceutical, such as a therapeutic protein, to changes in the drug's efficacy, safety, and long term stability (ESS). A great number of experimental methods are typically utilized to investigate the differences between forms of a macromolecule, yet conclusions regarding changes in ESS are frequently tentative.An opportunity exists, however, to relate changes in form to changes in ESS.At least once during the development of a new drug, a study is undertaken (at great expense) of the ESS of the drug upon perturbation by multiple manufacturing, formulation, storage and transportation variables. The data acquired is then used to build a model that relates changes in ESS to manufacturing, formulation, storage and transportation variables. It is not common in the pharmaceutical industry, however, to relate changes in comprehensive ESS data sets to comprehensive measurements of changes in macromolecular form.We bridge the gap between physical measurements of a macromolecule's form and measurements of its long term stability, utilizing two data sets collected in a collaboration between our group at the University of Kansas and a group at the Ludwig Maximilians University in Munich, Germany. The long term stability data, collected by the team in Germany, contains measurements of the chemical and conformation stability of Granulocyte Colony Stimulating Factor (GCSF) over a period of two years in 16 different liquid formulations. The short term iii physical data, collected in our lab, is comprised of spectroscopic characterization of the response of GCSF to thermal unfolding.The same 16 liquid formulations of GCSF were used in each study, allowing us to fit models predicting the long term stability of GCSF from short term measurements. We first apply a novel data reduction method to the short term data.This method selects data in the neighborhood of thermal unfolding transitions, and automates traditional comparative analyses. We then model the long term stability measurements using a linear technique, least squares fits, and a nonlinear one, radial basis function networks (RBFN). Using a Pearson correlation coefficient permutation test, we find that many of the fitted results have less than a 1% probability of occurring by chance.

The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine.