Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Microbial Cell Factories

Research

Johanna Panula-Perälä¹  Renata Rimšeliene²  Juozas Šiurkus²  Peter Neubauer³  Mario Kraft⁴  Uwe Horn⁴ 

[1] Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter Oulu, University of Oulu, PO Box 4300, FI-90014, Oulu, Finland;Fermentas UAB, V. Graiciuno 8, LT-02241, Vilnius, Lithuania;Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 71-76, D-1335, Berlin, Germany;Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter Oulu, University of Oulu, PO Box 4300, FI-90014, Oulu, Finland;Leibnitz Institute for Natural Product Research and Infection Biology, Beutenbergstr. 11a, D-07745, Jena, Germany;

关键词: Specific Growth Rate;    Shake Flask;    Mineral Salt Medium;    Ribosome Binding Site;    Luria Broth;   

DOI  :  10.1186/1475-2859-9-35

 received in 2010-03-23, accepted in 2010-05-20,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundBioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.ResultsHere, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.ConclusionsCultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.

【 授权许可】

Unknown   
© Šiurkus et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.