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Ernte von Influenza Virus aus tierischer Zellkultur : Infektionsverlauf, zentrifugale Klärung, Fällung verunreinigender DNA

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86366

Knöchlein,  A.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;
Univ. of Applied Sciences Hamburg, Faculty of Life Sciences;

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Zitation

Knöchlein, A. (2007). Ernte von Influenza Virus aus tierischer Zellkultur: Infektionsverlauf, zentrifugale Klärung, Fällung verunreinigender DNA. Master Thesis, Hochschule für Angewandte Wissenschaften, Hamburg.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-985F-5
Zusammenfassung
Increasing worldwide demand for influenza vaccines could lead to a bottleneck in supply in the near future. To satisfy the demand, process development of animal cell culture derived influenza vaccines is of growing importance. In this work a downstream processing scheme for the harvesting of cell-culture derived human influenza virus is presented. Preliminary investigations on the ideal time-point of harvest were carried out. The scheme for harvesting comprises centrifugal clarification for separation of cell debris, reduction of the impurity DNA by precipitation and removal of precipitates by microfiltration. For investigations on the ideal time-point of harvest, human influenza A virus A/PR/8/34 (H1N1) was propagated in roller bottles with serum-containing medium using MDCK cells as host. Independently of the multiplicity of infection, release of impurities (DNA and protein) followed the release of product with a time lag of approximately 7 hours. The time point of achieving maximum product concentration (based on HA and NA activity) was predominantly dependent on the inoculum lot. The ideal time-point of harvest was defined as the earliest time-point of maximum product concentration. Separation of cell debris was carried out in lab centrifuges. Independently of cell culture media (serum-free and serum-containing) or cultivation system (roller bottle or microcarrier cultivation) the maximum clarification efficiency achieved was 97 %, at a separator capacity of 1 • 10-8 m s-1. Maximal centrifugal clarification achieved was comparable to clarification by mircofiltration (0,45 µm). Dependent on cultivation media product recovery was 100 % on average for serum-containing and 82 % for serum-free media. Scale-up based on sigma-theory to a volume of 200 mL was possible. However, centrifugation needs to be carried out in conical vessels, since clarification efficiency was dependent on the vessel geometry. Aging of cultivation broth up to thirteen days did not show a strong impact on clarification performance. Furthermore, different cationic reagents (Ca2+, Mg2+, protamine sulphate, polyethylene­imine) were evaluated with respect to their selectivity of precipitating DNA from clarified cell culture supernatant. No selective DNA-removal could be achieved with polyvalent metal ions such as calcium and magnesium. Protamine sulphate-induced precipitation resulted in efficient clearance of DNA, but selectivity of product seemed to be strongly dependent on the initial concentration of virus. DNA was reduced to 3 % of the initial amount on average with a product recovery of 73 % (data of three batches from roller bottle cultivations). A polyethylenimine (PEI) concentration of 1 • 10-2 g (mg DNA)-1 reduced the amount of DNA to 0,24 µg mL-1, independently of the initial amount of DNA (data of four batches from microcarrier cultivations). Addition of polyethylenimine also led to reversible co-precipitation of product (based on NA activity) with virus being re-solubized at high PEI-concentrations (>10-2g PEI (µmol min-1)-1). Recovery of resolubized product seemed to be dependent on the initial amount and reached from 68…100 % (data of four batches from microcarrier cultivations). PEI precipitation was also carried out successfully with serum-free cultivation broth containing virus strains influenza A/Wisconsin/67/2005 (H3N2) and influenza B/Malaysia/2506/2004. The results of this work show, that harvesting of culture broth can be carried out at an optimal time-point. It can be concluded that centrifugal clarification in lab centrifuges is well suited for separating cell debris at different scales. DNA-precipitation using PEI was most promising, because selectivity was independent of the initial DNA concentration, virus strain and cultivation media. However, to ensure optimal precipitation conditions the influence of pH, ionic strength, temperature and molecular weight of the polymer need to be investigated.