(732a) Virus Purification By Aqueous Two Phase Extraction System

Authors: 
Heldt, C. L., Michigan Technological University
Vijayaragavan, K. S., Michigan Technological University



The prevention of diseases by vaccination is one of the most significant medical achievements and currently there is considerable interest in development of viral vaccines for human therapy. However, modest financial returns for vaccine businesses have been causing severe obstacles in new vaccine technology development. Additionally, urgencies and short timelines in downstream processing to establish scaled up clinical trials integrated with upstream processes demand a growing need for simple, easy scale up and cost effective technique. One such technique that is rarely applied to virus purification is aqueous two phase systems (ATPS). ATPS, consisting of a liquid-liquid biphasic system widely recognized for separation and purification of biomolecules like proteins and cell organelles, was first developed in Sweden by Huddleston in 1950’s. Partitioning of protein molecules using ATPS is a highly complex phenomenon due to hydrogen bonding, charge interactions, Van der Waal’s forces and surface hydrophobicity. We are currently using porcine parvovirus (PPV), a non-enveloped DNA virus of 18-26 nm diameter, as a model virus to develop vaccine purification techniques. The parvovirus particles are produced in porcine kidney cells (PK-13) containing fetal calf serum. Therefore, they contain serum proteins that can be immunogenic. We propose applying ATPS to effectively concentrate and purify PPV, primarily on the basis of hydrophobicity. Based on experimental studies, PPV has been recently validated by our laboratory to be more hydrophobic than typical blood and host cell proteins. For our ATPS experiments we have focused on polymer-citrate salt systems. We have successfully identified our ideal ATPS system after screening three PEG molecular weights and bulk salt concentrations ranging between 23- 41 w/w%. We report 98% virus recovery including both the top and interphase of the ideal ATPS system and removal of the serum proteins. The study presents a new technique for vaccine production and advance scientific understanding of viral purification strategies from extraneous contaminant proteins.