Denise Freimark

Bio: Denise Freimark is an academic researcher from University of Bayreuth. The author has contributed to research in topics: Chinese hamster ovary cell & Cell culture. The author has an hindex of 2, co-authored 2 publications receiving 16 citations.

A GFP-based method facilitates clonal selection of transfected CHO cells.

Abstract: The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.

Effect of process parameters and product-host-interaction on hVEGFA-production by recombinant Chinese hamster ovary cells.

Abstract: A potential producer clone was identified among recombinant, human vascular endothelial growth factor A (hVEGFA)-producing Chinese Hamster Ovary (CHO) K1 cells, using a recently established screening method. In batch spinner cultivations, the cells showed a maximum growth rate of 0.045 h−1, a final total cell density of 5.3 × 106 mL−1 (living cell density: 3.4 × 106 mL−1), and a final hVEGFA concentration of 207 μg L−1. Living cell density and productivity in the spinner cultivations could be increased by glutamine feeding. Transfer of the process to the bioreactor (batch mode, control of pH, T, and O2) resulted in a reduction of the growth rate by roughly 50%, while overall living cell density and productivity increased, largely due to an extension of the production phase. When the bioreactor was run in the fed-batch mode, growth rates were further reduced, while productivity and living cell densities reached a maximum (hVEGFA: 358 μg L−1, cells: 5.2 × 106 mL−1). In addition, the death rate of the hVEGFA-producing cells was considerably reduced compared with the parent cell line, most likely due to product-host-interaction. This hypothesis was corroborated when a second recombinant CHO cell line (antibody producer) was transfected with the hVEGFA gene and afterward consistently showed higher viable cell densities together with a significantly improved antibody titer. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

An 'omics approach towards CHO cell engineering

Abstract: Chinese hamster ovarian cells (CHO) cells have been extensively utilized for industrial production of bio- pharmaceutical products, such as monoclonal antibodies, human growth hormones, cytokines, and blood-products. Recent advances in recombinant DNA technology have resulted in the bioengineering of CHO cells that have robust gene amplification systems and can also be adapted to grow in suspension cultures. In parallel, recent advances in tech- niques and tools for decoding the CHO cell genome, tran- scriptome, proteome, and glycome have led to new areas of study for better understanding the metabolic pathways in CHO cells with the long-term goal of developing new biologics. This review paper discusses the recent advances in bioengineering strategies in CHO cell lines and the impact of the knowledge gained by CHO cell genomics, trans- criptomics, and glycomics on the future of CHO-cell engineering.

Towards dynamic metabolic flux analysis in CHO cell cultures

Abstract: Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.

Automated measurement and monitoring of bioprocesses: key elements of the M(3)C strategy.

Abstract: The state-of-routine monitoring items established in the bioprocess industry as well as some important state-of-the-art methods are briefly described and the potential pitfalls discussed. Among those are physical and chemical variables such as temperature, pressure, weight, volume, mass and volumetric flow rates, pH, redox potential, gas partial pressures in the liquid and molar fractions in the gas phase, infrared spectral analysis of the liquid phase, and calorimetry over an entire reactor. Classical as well as new optical versions are addressed. Biomass and bio-activity monitoring (as opposed to "measurement") via turbidity, permittivity, in situ microscopy, and fluorescence are critically analyzed. Some new(er) instrumental analytical tools, interfaced to bioprocesses, are explained. Among those are chromatographic methods, mass spectrometry, flow and sequential injection analyses, field flow fractionation, capillary electrophoresis, and flow cytometry. This chapter surveys the principles of monitoring rather than compiling instruments.

Measurement, monitoring, modelling and control of bioprocesses

Abstract: Automated Measurement and Monitoring of Bioprocesses: Key Elements of the M3C Strategy, by Bernhard Sonnleitner.- Automatic Control of Bioprocesses, by Marc Stanke, Bernd Hitzmann.- An Advanced Monitoring Platform for Rational Design of Recombinant Processes, by G. Striedner, K. Bayer.- Modelling Approaches for Bio-Manufacturing Operations, by Sunil Chhatre.- Extreme Scale-Down Approaches for Rapid Chromatography Column Design and Scale-Up During Bioprocess Development, by Sunil Chhatre.- Applying Mechanistic Models in Bioprocess Development, by Rita Lencastre Fernandes, Vijaya Krishna Bodla, Magnus Carlquist, Anna-Lena Heins, Anna Eliasson Lantz, Gurkan Sin and Krist V. Gernaey.- Multivariate Data Analysis for Advancing the Interpretation of Bioprocess.- Measurement and Monitoring Data, by Jarka Glassey.- Design of Pathway-Level Bioprocess Monitoring and Control Strategies Supported by Metabolic Networks, by Ines A. Isidro, Ana R. Ferreira, Joao J. Clemente, Antonio E. Cunha, Joao M. L. Dias, Rui Oliveira.- Knowledge Management and Process Monitoring of Pharmaceutical Processes in the Quality by Design Paradigm, by Anurag S Rathore, Anshuman Bansal, Jaspinder Hans.- The Choice of Suitable Online Analytical Techniques and Data Processing for Monitoring of Bioprocesses, by Ian Marison, Siobhan Hennessy, Roisin Foley, Moira Schuler, Senthilkumar Sivaprakasam, Brian Freeland.