Lectin-affinity chromatography for downstream processing of MDCK cell culture derived human influenza A viruses.
The presented study aims on the development of a capture step for the purification of cell culture derived influenza viruses using lectin affinity chromatography. Human influenza A/Puerto Rico/8/34 virus produced in Madin Darby canine kidney cells have been chosen as a model. The influenza A virus envelop possesses two viral glycoproteins: hemagglutinin and neuraminidase. Oligosaccharides of theses glycoproteins can be targeted as affinity ligands using specific lectins. First, lectins have been screened via lectin blots and spin columns. Adequate lectins have been chosen based on published glycan structures of hemagglutinin. The most specific binding was achieved via the galactose specific Erythrina cristagalli and Euonymus europaeus lectins. Second, the chromatographic separations characteristics of these lectins have been further determined via FPLC. These experiments revealed that the rate of hemagglutinin glycan binding to the ligands was higher with the E. europaeus compared to the E. cristagalli lectin. Third, viral recoveries in addition to the total protein and host cell DNA have been balanced in a series of E. europaeus lectin chromatography runs. The total protein and dsDNA content in the product fraction of the affinity chromatography was reduced from the starting conditions to 21% and 0.1%, respectively. The average viral recovery in the product fraction was 97%. SDS-PAGE analysis indicated that the majority of the eluted proteins were of viral origin. The reproducibility and column stability was confirmed in up to 25 runs applying six different virus product batches.
Opitz
Lars
L
Bioprocess Engineering, Max-Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany. opitz@mpi-magdeburg.mpg.de
Salaklang
Jatuporn
J
Büttner
Hermann
H
Reichl
Udo
U
Wolff
Michael W
MW
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004285:Dogs; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000302:isolation & purification; D009980:Influenza A virus / Q000302:isolation & purification* / Q000378:metabolism; D007252:Influenza Vaccines / Q000302:isolation & purification*; D037102:Lectins / Q000378:metabolism*; D009698:Nucleoproteins / Q000302:isolation & purification
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D037102:Lectins; D009698:Nucleoproteins