Dethardt Müller

MicroRNAs as targets for engineering of CHO cell factories

MicroRNAs (miRNAs) are strongly implicated in the global regulation of gene expression, and, in this regard, they consequently affect metabolic pathways on every regulatory level in different species. This characteristic makes miRNAs a promising target for cell engineering, and they could have multiple applications in medicine and biotechnology. However, a more profound, mechanistic understanding of miRNA action is needed for their potential to be translated into the development of industrially relevant cell factories with novel features. Here, we highlight the potential of miRNAs for the engineering of Chinese hamster ovary (CHO) cells, these being the most prevalent cell factory system for biopharmaceutical production. A key advantage of miRNAs, in contrast to most cell-engineering approaches that rely on overexpression of regulatory proteins, is that they do not compete for the translational machinery that is required to express the recombinant product. However, we also summarize the limitations and challenges that will have to be overcome to exploit fully miRNA technology.

Transcriptional profiling of phenotypically different Epo‐Fc expressing CHO clones by cross‐species microarray analysis

Chinese hamster ovary (CHO) cells exhibit large variabilities regarding growth, recombinant protein production and post‐translational processing during cell line development and clone selection. To accelerate the development of stable high quality cell factories, new efficient strategies for cell screening and clone selection are required. In our work, we combined phenotypic characterisation of recombinant CHO clones during early cell line development with transcription profile analysis using cross‐species microarrays. The objective was to identify genes or gene patterns that correlate with clone specific alterations in terms of productivity, sialylation capacity and stress resistance. In all high producer clones transcriptional profiling revealed a common enrichment of gene ontology categories related to protein metabolism, transcription, nucleus and nucleolus, whereas no common genes were differentially regulated in clones showing higher sialylation capacities. Furthermore, we identified predictive stress‐related marker genes that were up‐regulated in one clone without showing the corresponding phenotype at an early stage of development. Thus, we successfully applied gene expression profiling to allocate transcriptomal differences to specific phenotypes that changed during cell line development. These promising results will further increase our efforts to develop CHO specific microarrays that deliver information about the suitability of a clone candidate for industrial production.

Potentials and limitations of prokaryotic and eukaryotic expression systems for recombinant protein production – a comparative view

Background Within the last recent years biopharmaceutical sales have reached 30% of all new pharmaceutical sales in the United States expecting an increase from 30 billion USD (2003) to almost 60 billion USD until 2010 [1]. One major industry of the fast growing biopharmaceutical market is the manufacture of recombinant proteins for therapeutic and diagnostic use. Hence, the rising demand for new biopharmaceuticals requires increased production capacities as well as new production processes that exhibit increased space-time yields and shortened development times which also implies the use of suitable expression systems.

Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential

One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

CHARACTERISATION OF CHO SUBCLONES SHOWING PROFOUND CHANGES IN PERFORMANCE WHEN PROPAGATED IN DIFFERENT CULTIVATION SYSTEMS

Experiments were carried out with five serum-free adapted subclones of a recombinant CHO DHFR - line producing a monoclonal antibody. The bioreactor cultivation was performed in the multiple fermenter system Sixfors (Infors HT) in repeated batch mode. In the course of inoculum preparation cells were cultivated in T-flasks and spinner flasks (WV 0.125l and WV 0.5l). The General Cell Screening System (GCSS) was used as a tool for comparing growth characteristics of subclones derived from different cultivation systems in the course of inoculum preparation and bioreactor cultivation. It enables non-invasive determination of growth kinetics in 96 well plates [Steindl, 1990]. In addition, part of the inocula was used to perform batch cultivations in spinner flasks. The obtained results were compared with those found in the bioreactor. Cell concentrations were determined measuring the cell nuclei with a Coulter Counter after cell desintegration. Viability

Softmix, a new Scalable Mixing System for the Cultivation of Sensitive Cells on Microcarrier under Protein- Free Conditions

The Softmix is an equipment providing soft mixing conditions in animal cell bioreactors. It is based on an agitation mechanism totally different from conventional impeller technology (Fig. 1). The essential part is a perforated plate with tapered holes dipped into the liquid oscillating vertically at a controlled frequency and amplitude (Fig. 1). The main difference in comparison to an impeller is a more homogenous energy distribution within the mixing zone due to the fluid flow across the holes (Tab. 1). The oscillation generates a low speed current ideal for the cultivation of shear sensitive cells over a wide range of mixing conditions. Additionally, the system is equipped with a sieve for carrier retention and perfusion .

Comparison of Fluidized Bed and Fed Batch Reactor Cultures for Production of Anti-HIV-Antibody

High product yield, product quality and stable reactor culture conditions are a key to economically viable pharmaceutical bioprocesses. Two types of culture methods for production of recombinant anti-HIV-antibody were applied within this study. The tested cell line was a DHFR- recombinant CHO cell line expressing anti-HIV antibodies (CHO-4E10; Polymun Scientific, Vienna, Austria).Cells were cultured in a perfused lab-scale fluidized bed Cytopilot Mini™ reactor (Amersham Biosciences, Uppsala, Sweden) attached to macroporous Cytoline 1™ (Amersham Biosciences, Uppsala, Sweden) microcarriers and as suspension cells in a pilot scale stirred tank bioreactor fed-batch process. Both processes were performed using protein-free media. The systems were compared in terms of cell growth, product yield and product quality. Both systems were equally well suited for production of the recombinant protein in terms of product yield and product quality, however demonstrating an economic advantage of the fluidized bed process.

Continuous Perfusion versus Discontinuous Fed-Batch

The expression of recombinant glycoproteins that bear a cytotoxic potential is a major challenge using animal cells. Product yields in a range of 0.1 g/l to 1.0g/l as commonly reached for proteins that do not adversely affect the cultured cells are out of reach. The permanent expression of a cytotoxic protein at low specific productivities requires a bioprocess that allows the accumulation of product at concentrations adequate for purification. We compared a continuous perfusion process based on an ultrasonic cell retention device with a discontinuous fed-batch using a balanced nutrient concentrate. Theproduct yield in batch mode was set to a relative concentration of 1.0. In fed-batch the product yield was increased by 40% to a relative value of 1.4 accompanied by a significant loss of culture viability indicating the detrimental effect. However, under perfused conditions not only the volumetric productivity was increased as expected, but also the relative product concentration reached a valueof 6.0 at viabilities of more than 90%. Furthermore the product was harvested almost cell free at perfusion rates of 2d−1. Combining the perfusion mode (cell accumulation) and the fed-batch mode (product accumulation) the product yield was significandy increased toa relative value of 10.0 at final viabilities of still 65%. We thus developed a process capable of accumulating a cytotoxic protein in concentrations sufficient for downstream processing requirements.