While the ability to cary out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of biopharmaceuticals, a major obstacle to the implementation of displacement chromatography has been the lack of appropriate displacer compounds. It has recently been discovered that low molecular w eight (M.W.) dendrimers (480 dalton) can be successfully employed as displacers for protein purification in ion exchange systems. In addition to the fundamental interest generated by low M.W. displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers. The proposed research will expand upon this important result by addressing several critical issues relating tot he design of efficient low molecular weight displacers for protein purification by displacement chromatography. Specifically, our research will focus on the relationship between displacer chemistry and its efficacy as a protein displacer. The proposed research will include polymer synthesis, evaluation of the dynamic affinity and induced salt gradients and displacement experiments. A major challenge in the design of efficient low M.W. displacers is to develop molecules exhibiting sufficient affinity, while not producing inordinately high induced salt gradients. The synthetic work will include structural variants of the zeroth generation cationic dendrimer, anionic dendrimers, and various linear low molecular weight displacers. These synthesized compounds will be employed to carry out a detailed investigation of the interplay between displacer chemistry an important displacement chromatographic properties (i.e. dynamic affinity, induced gradients, and effective operating regimes). In addition, several operational issues such as ease of removal from contaminated zones and column regeneration will be studied. Finally, the low M.W. displacers developed in this research will be employed for the purification of proteins from complex biological mixtures. The proposed research will provide significant insight into the design of efficient low M.W. displacers for protein purification and will facilitate the implementation of this new technology for the purification of new therapeutic and diagnostic proteins at reduced costs. Furthermore, this work will facilitate the use of this high resolution/high throughput technique by medical researchers at the bench scale for the purification of novel biomolecules from dilute complex mixtures.
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