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High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells
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High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells
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Journal Article
High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells
2014
Overview
High cell densities in animal cell culture can be obtained by continuous perfusion of fresh culture medium across hollow fiber membranes that retain the cells. Careful selection of the membrane type and cut-off allows to control accumulation of target molecules and removal of low molecular weight compounds. In this report, perfusion with the scalable ATF (alternating tangential filtration, Refine Technology) system was evaluated for two suspension cell lines, the avian cell line AGE1.CR and the human cell line CAP. Both were cultivated in chemically defined media optimized for batch cell growth in a 1L stirred tank bioreactor connected to the smallest ATF unit (ATF2) and infected with cell line-adapted human influenza A virus (A/PR/8/34 (H1N1), typical diameter: 80–100nm).
At concentrations of about 25 million cells/mL three different membrane cut-offs (50kDa, 0.2μm and 0.5μm) were tested and compared to batch cultivations performed at 5 million cells/mL. For medium and large cut-offs no cell-density effect could be observed with cell-specific virus yields of 1428–1708 virions/AGE1.CR cell (infected with moi 0.001) and 1883–4086 virions/CAP cell (moi of 0.025) compared to 1292 virions/AGE1.CR cell and 3883 virions/CAP cell in batch cultures. Even at a concentration of 48 million AGE1.CR cells/mL (cut-off: 0.2μm) a cell-specific yield of 1266 virions/cell was reached. Only for the small cut-off (50kDa) used with AGE1.CR cells a decrease in cell-specific yield was measured with 518 virions/cell. Surprisingly, the ratio of infectious to total virions seemed to be increased in ATF compared to batch cultures. AGE1.CR cell-derived virus particles were present in the permeate (0.2 and 0.5μm cut-off), whereas CAP cell-derived virions were not, suggesting possible differences in morphology, aggregation or membrane properties of the virions released by the two cell lines.
To our knowledge, this is the first study that illustrates the potential of ATF-based perfusion of chemically defined media across cell-retaining membranes for production of an influenza A vaccine.
Publisher
Elsevier Ltd,Elsevier,Elsevier Limited
Subject
/ Animals
/ Batch Cell Culture Techniques
/ Batch versus perfusion culture
/ Biological and medical sciences
/ Birds
/ Cell Culture Techniques - methods
/ Fundamental and applied biological sciences. Psychology
/ Humans
/ Influenza A Virus, H1N1 Subtype - growth & development
/ vaccines
/ Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects)
/ virion
/ Virology
/ viruses
