This NSF award by the Chemical and Biological Separations program supports work by Professor Abraham Lenhoff at the University of Delaware to characterize quantitatively the structure-performance relationship in ion-exchange chromatography of proteins. The results of such analyses will then be used to develop systematic approaches for predicting chromatographic behavior on the basis of a small number of relevant experimental measurements. Ion-exchange chromatography is a major unit operation in industrial bioprocessing, and the prevalence of empirical screening methods can make efficient methods for reducing the scope of trial-and-error methods of optimization particularly valuable.

The proposed research will distinguish between traditional adsorbents on consolidated polymeric or gel-like base matrices, and polymer-modified phases in which additional flexible inclusions of various kinds have been shown to offer some performance improvements. For the traditional adsorbents, prior work has produced approaches to predict adsorption isotherms and transport properties to a degree that should allow effective prediction of overall chromatographic behavior. Such capabilities are still lacking for the polymer-modified adsorbents, however. We will therefore measure retention and transport properties for the polymer-modified adsorbents and seek to correlate them with structural characteristics of the materials. In addition, starting with traditional media and moving on to polymer-modified ones, mechanistic methods will be used to develop predictive models of protein loading and elution in packed columns.

The proposed research should have an impact in several areas. First, it should contribute significantly to improving the efficiency of conceptual and detailed design of chromatographic steps in bioprocessing. A complementary contribution will be to stationary-phase design, where suitable use of our results can allow desirable characteristics to be designed into such materials. Second, the research will be an effective educational vehicle for training of graduate and undergraduate students; numerous such students who have worked on previous projects of this kind are employed in the biotech and pharma industries. Finally, the proposed research will include a component to disseminate the findings to the larger community interested in chromatographic research and practice by making computational packages for column modeling available as both Matlab routines and as applets that can be run in any web browser.

Project Report

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. The workhorse of most purification processes is chromatography, in which small solid particles (adsorbent) packed into a tube (column) are used to bind (adsorb) proteins from a liquid medium comprising water with numerous substances dissolved in it. By then passing a clean medium through the column and manipulating its chemical composition, the different proteins adsorbed on the column can be removed sequentially, and the desired product then obtained in purified form. A major challenge in chromatography is to find the right balance between processing a lot of material (high throughput) and achieving adequate purity, and a critical element of this is to design and manufacture suitable adsorbent particles. One class of adsorbents involves particles with long-chain (polymer) molecules attached to them, with the protein binding then being facilitated by protein binding directly to the polymer, and protein entanglement in the polymer layer. The present project has investigated the mechanisms of protein binding to these polymer-modified adsorbents, based on measurement of a variety of properties of protein binding for proteins differing in size and in other important structural characteristics. These investigations have included column chromatography experiments as well as more specialized measurements, including the use of different kinds of microscopy, which have provided detailed information down to the level of molecules. The resulting outcomes include a better understanding of the behavior that can be expected for protein chromatography on polymer-modified adsorbents, including mathematical methods to predict certain aspects of behavior. These mathematical models can then be used in an engineering context to help choose suitable adsorbents for a given purification, to help design a column chromatography step, and to aid in design of new and improved adsorbent particles. The results obtained have been shared with practitioners at scientific conferences and in published articles, and we have collaborated with different manufacturers of adsorbents to help guide their development of improved materials.

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University of Delaware
United States
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