The objective of this research is to use measurements and mechanistic models of single and multicomponent protein solution thermodynamics as the basis for developing rational strategies for protein separation and purification. These strategies will build on the understanding of protein-protein interactions and of ways to manipulate and exploit them in designing effective separation pathways.
This research will employ numerous approaches developed in earlier grant periods, including: methods to measure protein interactions, such as self-interaction chromatography; methods to calculate protein-protein interactions, including electrostatic, dispersion and hydration interactions; and methods to calculate phase diagrams from complex, anisotropic potentials such as those that apply to proteins. A particular area of emphasis in the research will be to relate the nature of protein interactions to the resulting phase diagram, as well as to the types of amorphous dense phases observed in practice, such as precipitates and gels.
The project will support training of postdoctoral researchers and graduate students, as well as undergraduates. Such trainees in earlier grant periods have been successful in academic and industrial careers, and the undergraduates have been co-authors on numerous publications.
This project concerned the purification of proteins, primarily as part of the large-scale production of proteins for use as drugs; such protein-based pharmaceuticals represent a powerful class of therapeutic agents that have emerged in recent decades as a benefit of what is popularly referred to as genetic engineering. The proteins that are made by these methods are produced by specially developed cells that are grown in bioreactors, but to allow administration as drugs the protein product must be purified to separate it from other components produced by the cells. This separation process is a major challenge of modern biotechnology, and usually requires a number of sequential steps to guarantee adequate purity of the final product. In general proteins exist in an environment in which they are dissolved in water, but their "comfort level" in this milieu is also affected appreciably by other additives that may be present in solution; these include salts, acids or bases, sugars, as well as potentially other species. The "comfort level" is quantified in terms of the thermodynamic properties of the protein, which can be used to analyze and predict separations performance. Developing methods for doing this based on the structural properties of the protein as well as the solution composition was the central theme of this project. The methods that we developed were based on calculating the nature and strength of interaction of pairs of protein molecules, and on making a range of complementary measurements of either protein interactions or their consequences. One of the principal classes of consequences of interest is phase separation: if protein-protein interactions are strongly attractive, the protein molecules in solution can stick together and thereby form an extended aggregate structure, which may be manifested as a solid. This can take any of a number of forms, including crystals, gels or precipitates, and the formation of such assemblies can be used to aid in purification or in forming solids for other purposes; for example, insulin used by diabetics is usually administered in the form of small crystals, while formation of gels can be exploited in food manufacture. A particular class of proteins that we have investigated extensively is monoclonal antibodies. These molecules are designed based on the structure of the principal actors in the body's immune system, so they are the largest and fastest growing class of proteins used as therapeutics for a very wide variety of conditions and diseases. We have made measurements of the solution properties and the phase separation behavior of monoclonal antibodies received from several different pharmaceutical and biotechnology companies, and identified significant similarities but also interesting discrepancies among the different molecules. These results suggest that while very similar approaches can be adopted in processing many monoclonal antibodies, complications may ensue for molecules that deviate from the "normal" type of behavior. We are planning to pursue further the structural basis for the differences observed.
