Rationale Ansätze zur Prozessentwicklung eines Anti-EGF-IL2-Fusionsproteins mittels einer rekombinanten BHK-Zelllinie

摘要

The main goal of this thesis was to development a sufficient process strategy for the production of an anti-EGFR-IL2-fusionprotein with a recombinant baby hamster kidney cell line (BHK). The thesis is integrated within a cooperation project with MERCK KGaA, Darmstadt. The classical process development part includes the investigation of process parameters regarding cell growth and product formation. The second part describes the new rational aspects for process development with the tool of metabolic flux analysis in mammalian cells.1. Classical process developmentLimitations of the main nutrients (glucose, glutamine, amino acids) and inhibitions of metabolites (lactate, ammonia) as well as cultivation parameters (osmolality, pH-value and pO2 content, cultivation temperature) were investigated. Therefore a new parallel spinner cultivation system for suspension cells with controlled conditions was used (cellferm-pro from DASGIP AG, Juelich), Within a cooperation project with DASGIP the tool “metabolic activity monitoring” was developed, established and used for parallel batch cultivations and compared regarding growth and productivity within this thesis. The reduction of the cultivation temperature from 37,0°C to 35,5°C led to an increase of 60% of the space time yield and 20% improvement of the maximum specific product forming rate was obtained. This positive effect was transferred successful to 2L stirred tank reactor scale (perfusion with bleeding). Several cultivation and reactor concepts were compared concerning the suitability for the cultivation of the recombinant BHK-cells. Therefore chemostat and perfusion modes with different cell retention systems in stirred tank reactors were investigated (STY in chemostat: 1.7mg/(L*d), perfusion with internal spinfilter 200mg/(L*d) and with ultrasonic cell retention 82mg/(L*d)).2. Rational bioprocess developmentThe quantification of the intracellular fluxes in mammalian cells in stationary conditions (metabolic flux analysis) generates new tasks for rational bioprocess development. It was necessary to show the proof of concept for the investigation of intracellular metabolic fluxes in mammalian cells and to develop a metabolic model for BHK-cells. This resulted in successful cultivation system and mode for stationary metabolic flux analysis (chemosat in stirred tank reactor) with optimized flux rate (D=0,02h-1) obtained performing a X-D-Diagram. Preparation of samples for obtaining of the 13C enrichment in proteinogenic amino acids with 2D-NMR was established. BHK metabolic model with 99 constrains was developed with implementation of the balance data and the relative 13C-Isotopomere distribution results. Successful modelling and calculation of the intracellular metabolic fluxes with regards to the compartimentation and exchange fluxes resulted in consistent metabolic flux estimations (error residuum 252).3. Applications for metabolic flux analysisThe positive results of the investigation conditions for intracellular metabolic flux analysis for mammalian cells and the development and use of the BHK metabolic model allows to present the evidence of practical applications of this tool for bioprocess development in several examples. Exchange fluxes of the anaplerotic reactions were quantified. Tasks of metabolic engineering in BHK-cells were investigated with BHK-cells expressed cytoplasmatic recombinant Pryruvat-Carboxylase (GBF, Braunschweig). Intracellular metabolic fluxes were obtained and also presented. The investigation of nucleotide metabolism with example of the pyrimidine thymdin enabled the quantification of the intracellular exchange fluxes of the serin-glycine and the C1-metabolism. Limitation within the purin metabolism with example of the hypoxanthine was identified with the tool of metabolic flux analysis, so that an optimized supplementation of hypoxanthine was developed. The adaptation of different model variations identified the influences of the mitochondrial transport systems on intracellular metabolic fluxes. The metabolic flux analysis allowed the balance of the fatty acid synthesis. The tool of metabolic flux analysis identified limitations within the fatty acid synthesis. Experiments with supplementations of fatty acids in batch cultivations documented significant improvements of the central metabolism as well as an increase of the recombinant protein production (100% increase of the STY and 50% respectively 100% increase of the specific productivity).