Christoph Heinrich

Proteomic and metabolomic characterization of CHO DP-12 cell lines with different high passage histories.

Background For industrial pharmaceutical protein production fast growing, high producing and robust cell lines are required. To select more pH shift permissive and fast growing sub-populations, the CHO DP-12 (ATCC clone #1934) cell line, an anti-IL8 antibody producing CHO K1 (DHFR) clone, was continuously subcultured at high viability (>90 %) for more than four hundred days in shaking flasks using a chemically defined medium. During this long-term cultivation there was a repeated shift in pH and most robust and fast growing cells became accumulated [1]. Cell samples were cryopreserved at four different time points, after 21, 95, 165 and 420 days (in the following named sub-populations (SP)). The effects of long-term passaging before cryopreservation correlating with an increase in specific growth rate as well as changes in product formation and metabolism were examined in parallel bench-top bioreactor cultivation of SP21, SP95, SP165 and SP420 sub-populations. During exponential growth phase samples were taken for the analyses of differences in intracellular metabolites and protein expression (Please consider article “Growth characterization of CHO DP-12 cell lines with different high-passage histories” by Heinrich et al. in this issue for a detailed discussion of long-term cultivation, changes in specific growth rate, product formation and metabolic shifts).

Growth characterization of CHO DP-12 cell lines with different high passage histories

For industrial pharmaceutical protein production fast growing, high producing and robust cell lines are required. To select pH-shift permissive and faster growing sub-populations, the CHO DP-12 cell line was serially subcultured for more than four hundred days in shaker flasks. Initial adaptation to growth in suspension was carried out in chemically defined medium without hypoxanthine and thymidine (HT), while the final medium used for long term cultivation contains HT. Cell samples were cryopreserved at four different time points after 21, 95, 165 and 420 days. Cultivations of these four sub-populations (SP) in shaker flasks and bioreactors revealed considerable differences in specific growth rates and product formation as well as in the metabolism of glucose, lactate and several amino acids. For the elucidation of the intracelluar mechanism behind these alteration in growth characteristics and metabolism additional probes were analyzed using proteomic and metabolomic approaches [1].

Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI-MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.

Reconciling pillars of transient gene expression: From DNA prep via media, reagent and cell line development to holistic process optimization

Background Transient gene expression (TGE) is a multi-parametric process which is built upon four essential influencing pillars: First of all, it is imperative to have an easy to transfect cell line which allows high product titers and is cultivated in suspension [1]. Secondly, only a few of the commercially available cell culture media allow/support transient transfection and production. Thus, it is highly recommended to select the appropriate medium to grow and transfect the favored cell line [2]. Thirdly, the quality, source and backbone of the plasmid DNA which contains the genetic information for the protein of interest has a major impact [3]. Last but not least, to introduce plasmid DNA into mammalian cells, selection of a suited transfection reagent plays an important role for high-yielding TGE processes. In this work, we summarize our recent development of a novel TGE system for efficient transient transfection and expression in HEK cells. In cooperation with emp Biotech, InVivo BioTech Services developed a transfection reagent with very low cytotoxicity. A culture medium that can be used for transfection and production was designed in collaboration with Xell AG. The establishment of a TGE optimized HEK cell line (HEK-INV) and a method for large scale plasmid preparation of a corresponding vector complete the optimized production platform. It is easy applicable, scalable and supports large scale transfection for the production of gram quantities of IgG within a few days. Materials and methods Mammalian cells were cultivated and transfected in Xellvivo TM medium (Cat. No. 861-0001, Xell AG) under conditions of 37 °C, 5 % CO2 and 185 rpm agitation speed at 50 mm orbital diameter. For screening approaches 5x10E6 cells/mL were transfected with 2 pg DNA/cell and INVect transfection reagent (Cat. No. FK-0101-M001.0-001, emp Biotech GmbH) with INVect to DNA ratio of 6:1 (w/w) or 25 kDa L-PEI with PEI to DNA ration of 2:1 (w/w) in 8 mL culture volume in 50 mL bioreactor tubes. Expression of IgG1 was performed in 30 mL culture volume in 125 mL shake flasks or in 150 mL culture volume in 500 mL shake flasks respectively. Yields were quantified by proteinA affinity chromatography. Directed evolution was performed referring to Majors et al., 2009 [4]. In detail, an iterative process of evolution rounds followed by analysis of favorable attributes, cell selection and recovery was implemented. The corresponding flow cytometry analysis was performed using a Bio-Rad S3 cell sorter. Several E.coli strains and media were screened for high productivity, high quality and flexibility for DNA preparation in comparison to commercial kits in minipreparation scale. A purification process was implemented using a reusable and scalable anion exchanger. For large scale plasmid preparation, 6 L suspension was lysed, clarified and purified using an Äktaprime chromatography system. DoE was used for TGE process optimization after combining all developed elements.

Entwicklung von Zellkultur- und Feed-Medien

The development of culture media and supplements for fed-batch processes is one of the most complex steps in cell culture process development. We have developed chemically defined and animal component-free media and supplements for culturing Chinese Hamster Ovary cells (CHO) and hybridoma cells, improving the production of recombinant proteins.

Medium development beyond production media: Chemically defined media for transfection and single cell cultivation

Background State-of-the-art medium and feed development has proven its potential for increasing bioprocess efficiency many times. By the application of such advanced chemically defined and animal component-free formulations, in combination with recent cell lines, the yield of mammalian cell cultures was pushed to the binary gram per liter level. In this work, the current progress in designing special application media for transfection and single cell growth is presented. In the context of medium development, these techniques pose specific challenges which differ from production media. However, interest in such specialized products is high. On one side, clone selection is a key factor for robust and effective processes. On the other side, applications for transient transfection range from purposes in R&D, over the production of pre-clinical material to personalized medicine or vaccine production. In terms of upscaling, efficient transient gene expression (TGE) processes require a bifunctional medium supporting both cell growth and transfection.

Development and evaluation of a new, specially tailored CHO media platform

Background Today’s biopharmaceutical industry is under increasing pressure considering cost efficient development. Short timeframes rule the progress starting from the generation of producer cell lines to the establishment of a final production process. Hence, the timescale for optimization of cell culture media is small, but on the other hand it contains high potential for global process improvement. In this scope, our specially tailored media development platform, which allows a fast and reliable introduction of high-performance basis media and feeds, establishes new perspectives for an efficient process development.

Development of a chemically defined cultivation and transfection medium for HEK cell lines

Background In the process of generating a production cell, introduction of the gene of interest into the host cell can be performed by various physical, chemical or biological methods. Because of the greater scalability compared to physical methods and no safety concerns or restrictions that are associated with the use of viral systems, a transfection using chemical methods is of great interest. However, up to now up-scaling is limited by the challenge to transfect cells in conditioned media with the widely used reagent polyethylenimine (PEI). Considering the upscaling to gram yields, a culture medium that allows both, transfection and production is required. In this work, the current status in the development of such media supporting cell growth, transfection and protein production in HEK cells is presented. By this, processes will no longer be limited by media exchange prior transient transfection.

Introducing a new chemically defined medium and feed for hybridoma cell lines

Background Hybridoma technology was established in the 2nd half of the 20th century and in the view of current protein production it might seem old-fashioned. Despite, it is commonly used to produce monoclonal antibodies (mAbs) for R & D, clinical diagnostics or medical applications and the demand for mAbs produced by hybridomas is still high. However, compared to CHO, only a few serum-free hybridoma media are available and even less suppliers for chemically defined products are on the market. In this work, a new chemically defined medium and feed were developed to bring hybridoma processes to the next level and to target the existing gap in the market.