Jürgen Fieder
Boehringer Ingelheim
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Publication
Featured researches published by Jürgen Fieder.
Journal of Biotechnology | 2018
Sven Mathias; Simon Fischer; René Handrick; Jürgen Fieder; Patrick Schulz; Harald Bradl; Ingo Gorr; Martin Gamer; Kerstin Otte
With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity. Hence, we established a streamlined confocal microscopy-based methodology for CHO production cells investigating the distribution of the recombinant protein within the respective organelles of the secretory pathway and visualised the structure of the endoplasmic reticulum (ER) to be affected pinpointing towards an intra-ER bottleneck putatively hampering or limiting efficient secretion. The ER displayed not only a heavily altered morphology in comparison to a high immunoglobulin G (IgG)-producing cell line with a possibly inflated or overloaded structure, but the recombinant protein was also completely absent in the Golgi apparatus. Notably, the results obtained using an automated microscopy approach suggest the possible application of this methodology in cell line development and engineering.
Biotechnology Journal | 2017
Jürgen Fieder; Patrick Schulz; Ingo Gorr; Harald Bradl; Till Wenger
The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.
Archive | 2005
Kerstin Sautter; Jürgen Fieder; Ralf Dr. Otto; Barbara Enenkel
A major problem when establishing high-producing cell lines is the variability of integration sites. Integration of plasmid DNA into the genome of host cells occurs at random. As a consequence, selection of high producers is very time-consuming, costly and critical. Therefore, strategies which simplify and fasten the selection process are advantageous. Here, we present a system, that identifies and selects those of the transfected cells, which show the highest GFP-expression correlating with the expression of a product gene. This high expression system comprises expression vectors and serum-free transfection, selection and cultivation of CHO-DG44 cells.
Archive | 2003
Barbara Enenkel; Kerstin Sautter; Ralf Otto; Jürgen Fieder; Klaus Bergemann
Archive | 2004
Barbara Enenkel; Jürgen Fieder; Ralf Dr. Otto; Thomas Krieg
Journal of Biotechnology | 2005
Jürgen Fieder; Nina Wagner; Stefanos Grammatikos; Helmut Hoffmann; Hitto Kaufmann; Ralf Otto
Archive | 2004
Barbara Enenkel; Jürgen Fieder; Ralf Dr. Otto; Thomas Krieg
Archive | 2009
Rebecca Bischoff; Jürgen Fieder; Hitto Kaufmann; Thomas Krieg
Archive | 2007
Barbara Enenkel; Kerstin Sautter; Jürgen Fieder; Ralf Otto; Klaus Bergemann
Archive | 2004
Barbara Enenkel; Kerstin Sautter; Jürgen Fieder; Ralf Otto; Klaus Bergemann