Protein and Viral Partitioning in
Two-Phase Aqueous Micellar systems

Professor Daniel Blankschtein
Department of Chemical Engineering
Massachusetts Institute of Technology

Date: Thursday, Oct 30, 2003
Time: 4:00 p.m.
Place: Engineering II, Room 3361

ABSTRACT

Several biotechnological applications require the separation of proteins from viruses. These include large-scale applications, such as viral clearance, production of gene delivery viral vectors, and vaccine production, as well as small-scale applications, such as preparative biochemistry and diagnostics. Accordingly, a readily scalable separation process is desirable. Although liquid-liquid extraction is scalable, conventional oil-water solvents cannot be used because nonpolar solvents denature biomolecules. In this presentation, I will summarize our recent experimental and theoretical efforts in utilizing two-phase aqueous micellar systems to carry out the liquid-liquid extraction, where the coexisting micelle-rich and micelle-poor phases consist predominantly of water, and can therefore provide a friendly environment to biomolecules.

Studies were conducted to identify the fundamental mechanisms responsible for the observed viral partitioning behavior in the two-phase aqueous nonionic C10E4 micellar system. Competitive inhibition and partitioning studies demonstrated that possible attractive interactions between the C10E4 micelles and the tailspikes/capsids of bacteriophage P22 (a model virus) were not significant. However, other experiments along with the development of a new theory of viral partitioning showed that the primary mechanisms governing viral partitioning are entrainment of micelle-poor domains in the top, micelle-rich phase and excluded-volume interactions between the viruses and the C10E4 micelles.

Methods for improving the separation of proteins from viruses were also identified. The volume ratio was varied to increase the yield of lysozyme (a model protein) in the top, micelle-rich phase while maintaining a high yield of bacteriophage P22 in the bottom, micelle-poor phase. It was also found that mixed (nonionic/anionic) micellar systems could be used to modulate both excluded-volume and electrostatic interactions between the proteins and the mixed micelles. In particular, the addition of the anionic surfactant SDS to the C10E4 micellar system improved the separation of lysozyme from bacteriophage P22. A molecule-thermodynamic theory of protein partitioning in two-phase aqueous mixed (nonionic/anionic) micellar systems was developed, and found to provide reasonable quantitative predictions of the observed protein partitioning behavior in the mixed micellar system examined.