NMR Used to Purify Proteins, Improving Drug Development

This is especially true in the case of drugs that utilize proteins, which are very difficult to separate from other potentially deadly impurities. Scientists are now using nuclear magnetic resonance (NMR) to understand and improve an important protein purification process.

"We hope to use our insights to help those in the industry develop improved processes to provide much less expensive drugs and dramatically reduce healthcare costs,” said study author and professor of polymer engineering Dr. Steven Cramer of the Center for Biotechnology and Interdisciplinary Studies (CBIS) at Rensselaer Polytechnic Institute (Troy, NY, USA). The study's findings were published in the September 2, 2010, online early edition of the journal Proceedings of the [U.S.] National Academy of Sciences (PNAS).

The process of multimodal chromatography has recently generated significant interest in the pharmaceutical industry. At its most basic, this process separates proteins from their surrounding materials, such as DNA and other proteins. The process works by encouraging the desired protein to stick to a material that contains a ligand, a type of molecular glue. Each ligand is only attracted to specific parts of certain proteins. Having been separated from the mixture, the specific protein can now be obtained in purer form, facilitating its eventual use as a biotherapeutic.

The more selective the ligand is at binding to a specific protein, the more effective the process is, and the less additional steps are required to produce the ultimate drug. This results in reduced costs for the production of the drug. But in spite of its widespread use and benefits, there is very little understood about how the process actually works or how the ligands can be enhanced. "We are trying to understand what exactly is making these materials so useful for separating proteins,” Dr. Cramer commented. "And what we are looking to uncover are the fundamental interactions within the chromatographic process that make the separations possible and efficient.”

For this study, the researchers used several of the advanced research facilities within CBIS. Using the microbiology and fermentation core, Dr. Cramer and his colleagues grew several mutants of a protein called ubiquitin. This group of modified proteins is referred to as a protein library.

To compare the difference between multimodal systems and more conventional chromatography, the investigators ran the library through a less sophisticated chromatography system called ion exchange chromatography, as well as the multimodal system. They found that there was very little to no difference in the binding of proteins to ligands in the traditional ion exchange system. In contrast, there were huge fluctuations in the binding of some of the different mutants within the multimodal system.

To study further into why this occured, the scientists input ubiquitin and the multimodal ligands into the huge 800 MHz NMR machine at Rensselaer's CBIS. The NMR utilizes magnetic properties within organic materials to provide data on the minuscule molecular chemical properties of the material. From the NMR data, they were able to determine what part and type of the protein the ligands were binding and the strength with which they would bind. Their findings confirmed the previous multimodal chromatography comparison experiments, demonstrating that each of the protein mutants that strongly fluctuated in their binding strength in the multimodal chromatographic system were also the same ones identified with the NMR.

"This research is helping us develop a fundamental understanding of selectivity,” Dr. Cramer said. Working with his team, Dr. Cramer will work to design improved ligands and improved processes for their purification.

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Center for Biotechnology and Interdisciplinary Studies at Rensselaer Polytechnic Institute