Purification of porcine reproductive and respiratory syndrome virus from cell culture using ultrafiltration and heparin affinity chromatography.
Porcine reproductive and respiratory syndrome (PRRS) virus is the causative agent of the most significant infectious disease currently affecting the swine industry worldwide. Density gradient ultracentrifugation remains the most commonly used method for porcine reproductive and respiratory syndrome virus (PRRSV) purification. However, this technique has notable drawbacks including long processing time and limited processing volume in each run. To overcome these limitations, a scalable process was developed. PRRSV propagated in MARC-145 was released by three freeze/thaw cycles. After a low speed centrifugation step, the virus particles in the supernatant were concentrated twice by an ultrafiltration step. The ultrafiltration step concentrated the virions effectively with no detectable loss while some cultural/cellular proteins were removed. The virions in the ultrafiltration retentate were then applied to a heparin affinity column on a fast performance liquid chromatography unit. The combined ultrafiltration and heparin affinity chromatography process removed more than 96% of cellular and medium proteins. During a stepwise elution strategy, the viral particles were eluted at two separate peaks recovering 27.5% and 25.4% of viral particles loaded onto the column with a purity of 194 and 3917 particles/microg protein, respectively.
Hu
Jianzhong
J
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 200 Seitz Hall, Blacksburg, VA 24061, USA.
Ni
Yanyan
Y
Dryman
Barbara A
BA
Meng
X J
XJ
Zhang
Chenming
C
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D006493:Heparin / Q000378:metabolism; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D012965:Sodium Chloride; D013552:Swine; D014325:Tromethamine; D014462:Ultrafiltration / Q000379:methods*; D014771:Virion / Q000302:isolation & purification* / Q000378:metabolism; D014776:Virus Cultivation / Q000379:methods*
D014325:Tromethamine; D012965:Sodium Chloride; D006493:Heparin