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New purification process joins high throughput with high selectivity

05/31/2005

Penn State chemical engineers have demonstrated proof of concept for a new protein purification process that combines ultrafiltration's high throughput with high specificity achievable through electrically-charged dyes that bind to the protein.

Ultrafiltration is widely used now in the pharmaceutical industry, by milk and whey producers and in water purification. The new process promises to broaden the scope of ultrafiltration to more fine separations.

In the proof of concept experiments, the protein of interest was tagged with a small, commercially-available, negatively-charged dye molecule that can be easily removed. When the solution to be purified flowed through a negatively-charged ultrafiltration membrane, the protein of interest, now negatively-charged because of the attached dye, was retained in higher proportion than when it wasn't tagged.

Dr. Andrew Zydney, professor of chemical engineering and a developer of the process, says, "Classically, in ultrafiltration, the size of the pores in the filter determined what could get through. Recent studies have demonstrated that additional retention can be achieved using electrically charged membranes if the protein were of like charge. However, these new experiments have shown that you can enhance retention for the same size pore by attaching a charged dye marker to the protein of interest to change its electrical charge."

The new purification process is detailed in a paper, "Controlling Protein Transport in Ultrafiltration Using Small Charged Ligands," published online in the current issue of the journal, Biotechnology and Bioengineering. The authors are Suma Rao, doctoral candidate in chemical engineering, and Zydney, who is also head of the Department of Chemical Engineering.

For their proof of concept experiments, the researchers chose bovine serum albumin (BSA) and the dye Cibacron Blue. They obtained and compared sieving data from neutral and negatively-charged filters. They found that the addition of about one gram per liter of Cibacron Blue to an eight gram per liter solution of BSA caused a reduction in the BSA sieving coefficient by more than 100 times (two orders of magnitude) when passed through the negatively-charged filter.

The researchers note in their paper that it would be possible to design a system in which the protein and dye were separated after filtering and the dye recycled for subsequent processes. For example, in the case of Cibacron Blue and BSA, decreasing the acidity of the solution and adding certain salts causes the dye to detach readily from the BSA. The dye can then be recovered through filtration.

While the proof of concept experiments were conducted with just one protein, the researchers say that it should be possible to exploit the specificity of dye-binding interactions to enhance the selectivity of other protein separations providing new opportunities for membrane systems.