Propagation of influenza virus in Vero cells using Cytodex TM microcarriers in WAVE Bioreactor TM systems

Transcription

1 Propagation of influenza virus in Vero cells using Cytodex TM microcarriers in WAVE Bioreactor TM systems Ann-Christin Magnusson, Therese Lundström, *Christian Kaisermayer and Mats Lundgren GE Healthcare Bio-Sciences AB, Björkgatan 30, SE Uppsala, Sweden * GE Healthcare Europe GmbH, c/o Institute of Applied Microbiology Muthgasse 18, Haus B A-1190 Wien First published at the 241st ACS spring meeting, Anaheim, USA, March 2011

2 Introduction The use of cell culture-based Live Attenuated Influenza Vaccines (LAIV) is seen as one way to secure a rapid supply of a large number of influenza vaccine doses. This could be critical in case of a pandemic outbreak. Thus, it is important to reduce the time of vaccine development and to reach the market rapidly. For example, disposable bioreactors will be useful in production of high quality material for clinical trials. 2 /Propagation of influenza virus in Vero cells

3 Introduction We have performed a study on propagation of live influenza virus in Vero cells by using Cytodex microcarriers in WAVE Bioreactor systems. Different cell culture media and supplements have been tested. The data show that production of influenza virus in WAVE Bioreactor systems is a fast and convenient alternative to conventional bioreactors and could be a good foundation for further scale-up. 3 /Propagation of influenza virus in Vero cells

4 Material & Methods WAVE Bioreactor / 10 L Cellbag TM (20/50) / (GE Healthcare) Cytodex 1 (GE Healthcare) 3 g/l Animal-component-free medium (OptiPRO-SFM, Invitrogen) Vero cells (ECACC) Supplements: Pluronic F-68, Soy Peptone and Trypsin inhibitor Cell density at inoculation : ~ 0.4x10 6 cells /ml Cell density at infection: ~1.0x10 6 cells /ml Agitation: First 24 h post inoculation: continuous rocking 12 rpm / 5º After 24 h: continuous rocking 14 rpm / 5º Tissue culture infectious dose 50 assay (TCID 50 ) Hemagglutinin (HA) measured with Biacore TM assay 4 /Propagation of influenza virus in Vero cells

5 Material & Methods Virus stock preparation: Influenza A/Solomon Islands/3/2006 (H1N1) IVR145 WHO (egg derived) was adapted through a number of passages in Vero cells. A working virus bank was created and tested for infectivity by TCID 50 analysis (Reed & Muench). 5 /Propagation of influenza virus in Vero cells

6 Different Cell Culture Media Cell attachment and growth on Cytodex 1. A clear difference can be seen after 48 h inoculation. 6 /Propagation of influenza virus in Vero cells

7 The effect of supplements Cytodex 1 was used for expansion of Vero cells in animal component free conditions. Supplementation with peptones was required for robust cell growth. 7 /Propagation of influenza virus in Vero cells

8 Kinetics of HA and TCID 50 During Culture Samples were taken at different time points post infection. The concentration of hemagglutinin (HA) was measured with a Biacore assay and the infectious titer was analyzed with TCID 50. Infectivity peaks relatively early whereas HA concentration increases slowly for several days. Thus, it may be important to harvest early in a LAIV process. 8 /Propagation of influenza virus in Vero cells

9 Influenza Virus Production Workflow in WAVE Bioreactor 9 /Propagation of influenza virus in Vero cells

10 TCID 50 from Different 2 L Influenza Batches 10 /Propagation of influenza virus in Vero cells

11 Conclusion Here we show a method that we have developed for the propagation of influenza A virus in Vero cells using Cytodex microcarriers in WAVE Bioreactor system. Conditions for cell attachment in terms of mixing conditions has to be optimized. Additives such as Pluronic F68 as shear protectant and Soy Peptone are required in serum-free conditions. A substantial number of LAIV vaccine doses can be produced in disposable Cellbag. The WAVE Bioreactor system is a suitable system for propagation of influenza virus using adherent Vero cells on Cytodex microcarriers in an animal component free process. 11 /Propagation of influenza virus in Vero cells

12 Acknowledgments For contact information for your local office, GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE, imagination at work and GE monogram are trademarks of General Electric Company. Biacore, Cytodex, Cellbag and WAVE Bioreactor are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published March 2011 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. 12 /Propagation of influenza virus in Vero cells