(15f) Virus Purification by Aqueous Two Phase Extraction System

AIChE 2012 Annual Meeting, Pittsburg, PA October 28-November 2, 2012

Division: Bioengineering

Group: Bioseparations and Downstream Processing

 

Virus Purification by Aqueous Two Phase Extraction System

K. SAAGAR VIJAYARAGAVAN (kskhrupa@mtu.edu) & CARYN L. HELDT(heldt@mtu.edu)

Department of Chemical Engineering, MTU

 

Rising social concerns over potential viral contamination and infection from blood plasma derivatives in the last few decades have raised the need of biotheraputic safety measures for downstream processes in the biopharmaceutical industry. One strong technique that is often applied to protein purification, but rarely to virus removal is aqueous two phase systems (ATPS). ATPS was first developed in Sweden by Huddleston in 1950’s and consists of a liquid-liquid biphasic system widely recognized for separation and purification of biomolecules, like proteins and cell organelles. Due to its ecofriendly, economic, and easy scale up, it is possible to implement this separation system to different biotechnological applications. The partitioning of protein molecules into different aqueous phases is a highly complex phenomenon due to hydrogen bonding, charge interactions, Van der Waal’s forces and surface hydrophobicity. Porcine Parvovirus (PPV), a non-enveloped DNA virus of 18-26 nm diameter, is a model virus used to test virus removal.  It is a model of the human B19 parvovirus, which has been reported to contaminate biotheraputic products. PPV has been studied and recently validated to be more hydrophobic than typical blood and host cell proteins, by our laboratory. Based on this experimental data, we are proposing ATPS to effectively separate PPV to one of the liquid-liquid immiscible phases due largely on hydrophobicity.  In our ATPS experiments we use both a polymer-polymer and polymer-salt system. We are currently focusing our efforts on a polymer-salt system comprising of PEG and three different salts, namely phosphate, sulphate and citrate. We have successfully identified the phase separation based on binodial phase curve lines and are using Alexa fluor-tagged PPV to determine the separation of the virus in individual phases of the biphasic system.  We have successfully separated PPV with this system. Some of the factors affecting partition coefficient (K) are pH, neutral salts addition, salt concentration, PEG molecular weight and PEG concentration. The study will provide scientific understanding into viral clearance strategies and improve biotheraputic safety in manufacturing processes.