Harvesting and concentration of human influenza A virus produced in serum-free mammalian cell culture for the production of vaccines.
A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 microL)(-1) and turbidities of 0.479 OD measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 microg/mL and 5.73 microg/mL, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fiber modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 L/(m(2) h) and a wall shear rate of 9,500 s(-1). The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.
Kalbfuss
Bernd
B
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, Magdeburg 39106, Germany. kalbfuss@mpi-magdeburg.mpg.de
Genzel
Yvonne
Y
Wolff
Michael
M
Zimmermann
Anke
A
Morenweiser
Robert
R
Reichl
Udo
U
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000254:growth & development* / Q000302:isolation & purification*; D007252:Influenza Vaccines / Q000096:biosynthesis*; D007251:Influenza, Human / Q000517:prevention & control*; D007668:Kidney / Q000821:virology*; D014776:Virus Cultivation / Q000379:methods*
D016895:Culture Media, Serum-Free; D007252:Influenza Vaccines