Researcher

Alexander Rath
Phone:+49 391 6110 167
Email:rath@...

Sandtorstrasse 1, 39106 Magdeburg, Germany

Susann Freund

Additional Information

Collaborations:

Prof. Dr. Thomas Noll, Institute of Cell Culture Technology (University of Bielefeld).

Prof. Dr. rer. nat. An-Ping Zeng, Institute of Bioprocess and Biosystems Engineering (University of Hamburg-Harburg).

Prof. Dipl. Ing. Dr. tech. Elmar Heinzle, Research Group Biochemical Engineering (University of Saarbruecken).

ProBioGen AG


Funded by BMBF-initiative FORSYS
SysLogics Workpackage 3 - Metabolom and Proteom Analysis

Start:
2008/07/03

End: April 2013

Process Characterisation and Optimisation for a Recombinant Protein Produced by a Novel Human Designer Cell Line

Process Characterisation and Optimisation for a Recombinant Protein Produced by a Novel Human Designer Cell Line

Motivation

The dynamics of the central metabolism and cell cycle of two novel suspension cell lines (AGE1.HN and AGE1.HN.AAT) both derived from human neural tissue and developed by an industrial partner (ProBioGen AG, Berlin, Germany) are under investigation by the SysLogics consortium funded by the BMBF (Forsys Partners). Within this consortium, we are focusing on the characterization of cell growth, metabolism and recombinant protein production of designer cell lines during batch and continuous cultivations under different cultivation conditions. Systems biological approaches including dynamic modeling of cell growth and central metabolism are used to uncover limiting process factors, to predict cell response to different cultivation conditions and to support identification of process parameters crucial for product yields.

Work strategy

  • Batch cultivations to determine kinetics and parameters for process control and modelling
  • Continuous cultivations under substrate limitation for analysis of different process perturbations
Figure 1: Scheme of continuous cultivation experiments Zoom Image
Figure 1: Scheme of continuous cultivation experiments

Determination of intracellular metabolites of the central metabolism

A method established by Ritter [1,2] for adherent cells is being adapted to analysis of suspension cells:

  • LC-MS system (Dionex, DX320) with conductivity and UV channel and quadrupole mass spectrometer (electrospray ionization) [1]
  • About 30 different metabolites from glycolysis, TCA cycle and and nucleotides from energy metabolism quantifiable [2]
  • Fast quenching of cell metabolism of adherent cells growing in 6 well plates [2]

Todo:

  • Removal of supernatant is time consuming for suspension cells
  • Therefore, further development of method for analysis of intracellular metabolites extracted from suspension cells with focus on quenching of cell metabolism
Figure 2: LC-MS system (Dionex, DX320) for analysis of intracellular metabolites (A) and detected metabolite peaks in the UV channel (B) Zoom Image
Figure 2: LC-MS system (Dionex, DX320) for analysis of intracellular metabolites (A) and detected metabolite peaks in the UV channel (B) [less]

References

[1] Ritter, J. B.; Genzel, Y. & Reichl, U. "High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture." Journal of Chromatography, 2006, 843: 216-226.

[2] Ritter, J. B.; Genzel, Y. & Reichl, U. "Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: Optimization using experimental design." Analytical Biochemistry, 2008, 373: 349-369.

Related projects

Metabolic Profiling in Mammalian Cells
Enzymatic Characterization of Mammalian Cells
SysLogics - Modelling of Central Metabolism

 
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