(506b) Virus Flocculation in the Presence of Protecting Osmolytes

Virus Flocculation in the Presence of Protecting Osmolytes

Maria F. Tafur, Eric Pearson & Caryn L. Heldt

Department of Chemical Engineering, Michigan Technological University, 1400 Townsend Drive, Houghton, MI, 49931 USA

Viruses are best known for their ability to cause disease.  However, viruses can be manipulated to become vaccines or engineered to deliver genetic material that help combat disease.  There is a need to improve the purification of viruses for the production of therapeutic virus particles.  The main unit operation for virus purification is chromatography.  There are many disadvantages to chromatography, including the need for high product concentration, large solvent quantities, and the low yield of large particles.  We have studied the use of protecting osmolytes in the flocculation of viruses for improved purification.  Osmolytes are natural, organic compounds that stabilize intracellular proteins against environmental stresses, such as severe temperature or high osmotic pressure, by changing the water content of cells.  Protecting osmolytes have the ability to fold proteins by structuring water around the protein surface, which can either stabilize or destabilize the protein.  We are currently working with porcine parvovirus (PPV), a non-enveloped, icosahedral, single-strand DNA virus, to demonstrate the ability of osmolytes to flocculate viruses.  The structuring of water around the hydrophobic virus is proposed as the method for PPV flocculation.  PPV was flocculated with different concentrations of osmolytes, followed by filtration through a 0.20 µm filter.  This large pore-sized filter is usually used to retain bacteria, not small viruses.  Using high throughput screening, we have discovered that protecting osmolytes flocculate PPV and demonstrate a high removal (80-97%) with the large pore-sized filter.  The virus aggregate particles are very fragile and can be broken with high agitation.  When the pH of the osmolytes solutions was adjusted close to the isoelectric point of the virus, improved virus removal was achieved.  We propose to use protecting osmolyte flocculation as a potential platform purification approach for vaccines and gene therapy vectors.