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Purification of cell culture-derived human influenza A virus by size-exclusion and anion-exchange chromatography.

Authors
  • Kalbfuss, Bernd1
  • Wolff, Michael
  • Morenweiser, Robert
  • Reichl, Udo
  • 1 Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany. [email protected] , (Germany)
Type
Published Article
Journal
Biotechnology and bioengineering
Publication Date
Apr 01, 2007
Volume
96
Issue
5
Pages
932–944
Identifiers
PMID: 16937411
Source
Medline
Language
English
License
Unknown

Abstract

A process comprising of size-exclusion chromatography (SEC) and anion-exchange chromatography (AEC) was investigated for downstream processing of cell culture-derived influenza A virus. Human influenza virus A/PR/8/34 (H1N1) was propagated in serum-free medium using MDCK cells as a host. Concentrates of the virus were prepared from clarified and inactivated cell culture supernatants by cross-flow ultrafiltration as described before. SEC on Sepharose 4 FF resulted in average product yields of 85% based on hemagglutination (HA) activity. Productivity was maximized to 0.15 column volumes (cv) of concentrate per hour yielding a reduction in total protein and host cell DNA (hcDNA) to 35 and 34%, respectively. AEC on Sepharose Q XL was used to separate hcDNA from virus at a salt concentration of 0.65 M sodium chloride. Product yields >80% were achieved for loads >160 kHAU/mL of resin. The reduction in hcDNA was 67-fold. Split peak elution and bimodal particle volume distributions suggested aggregation of virions. Co-elution with hcDNA and constant amounts of hcDNA per dose indiciated association of virions to hcDNA. An overall product yield of 52% was achieved. Total protein was reduced more than 19-fold; hcDNA more than 500-fold by the process. Estimation of the dose volume from HA activity predicted a protein content at the limit for human vaccines. Reduction of hcDNA was found insufficient (about 500 ng per dose) requiring further optimization of AEC or additional purification steps. All operations were selected to be scalable and independent of the virus strain rendering the process suitable for vaccine production. (c) 2006 Wiley Periodicals, Inc.

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