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Harvesting and concentration of human influenza A virus produced in serum‐free mammalian cell culture for the production of vaccines
Author(s) -
Kalbfuss Bernd,
Genzel Yvonne,
Wolff Michael,
Zimmermann Anke,
Morenweiser Robert,
Reichl Udo
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21139
Subject(s) - ultrafiltration (renal) , chromatography , chemistry , virus , microfiltration , hemagglutination , agarose gel electrophoresis , yield (engineering) , biochemistry , biology , membrane , dna , virology , materials science , metallurgy
Abstract 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 β‐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 µL) −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 µg/mL and 5.73 µg/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. Biotechnol. Bioeng. 2007;97:73–85. © 2006 Wiley Periodicals, Inc.