Publications de Florian Maria Wurm

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Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (G ...

Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a mo

In the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venou

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one enco

We have characterized sequences expressed in Chinese hamster ovary (CHO) cells which are related to the intracisternal A-particle (IAP) genes of mice and Syrian hamsters. Several cDNA clones homologous to Syrian hamster IAP probes have been isolated and used to evaluate the abundance and expression of these retroviruslike sequences. DNA blot analysis with homologous Chinese hamster IAP probes revealed that IAP-related sequences are present in CHO cell DNA at moderately repetitive levels (approximately 300 c

Recombinant therapeutic proteins represent a major class of biopharmaceuticals. They are produced in various hosts including bacteria, yeast, cultivated mammalian and plant cells, and transgenic plants and animals. Chinese hamster ovary (CHO) cells are the most widely used mammalian host for their production. Currently, about one-third of all approved recombinant therapeutic proteins are produced in this cell line. This article is a summary of the molecular and cellular processes involved in therapeutic pro

Various methods exist to transfect mammalian cells in culture. It is generally accepted that individual methods have to be optimized for each of the cell lines or cell types used. Despitethe use of optimized protocols, significant day-to-day variationsin transfection efficiency regularly occur. We postulate that the;status' of cell populations prior to transfection is involved insuch variability. This study evaluates standardized transfectionsdone at different phases of the cell cycle. Cell synchronizationw

Disposable cylindrical shaken bioreactors using plastic bags or vessels represent a promising alternative to stainless steel bioreactors, because they are flexible, cost-effective and can be pre-sterilized. Unlike conventional well-established steel bioreactors, however, such disposable bioreactor systems have not yet been precisely characterized. Thus, the aim of this current work is to introduce a new power input correlation as a potential means to characterize the hydrodynamics of these new systems. A se

DNA fragments encoding structural information of the Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene were cloned into pUC plasmids [Vieira and Messing, Gene 19 (1982) 259-268]. None of the hybrid plasmids were able to direct the synthesis of significant amounts of gC related peptides. Several of the plasmid-bearing strains, however, exhibited inhibition characteristics which can be correlated with the presence on the plasmid of specific gC gene sequences. After insertion of gC DNA fragments int

Orbitally shaken bioreactors are an emerging alternative to stirred-tank bioreactors for large-scale mammalian cell culture, but their fluid dynamics is still not well defined. Among the theoretical and practical issues that remain to be resolved, the char ...

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are ...

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.

Antibody therapeutics have demonstrated transformative impacts on improving the quality of life of millions of patients, whereas advances in antibody discovery technologies have imposed a significant production challenge for the generation of a large diversity of therapeutic antibody candidates. A demand for the rapid production of dozens of purified antibodies in 10-mg quantities is entailed for functional screening and molecular assessment studies. Here, we present a robust semi-automated production proto

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in t

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient ge ...

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estim ...

This study correlates the fluorescent signal from stable recombinant CHO cell lines expressing the green fluorescent protein (GFP) at high levels with biomass or cell number, extending the use of fluorescent proteins to applications and assays where cell growth rates are important. Using a standard fluorometer, growth of these cells can be quantified noninvasively in multiwell plates, and because signals are obtained without preparation, the same culture samples can be measured repeatedly. Even with a small

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-mL tubes with a ventilated cap (OSR50), origina ...

Fluorescent protein expressing cells with very high fluorescence are described as well as a method to obtain such cells. The fluorescence of such cells can be as high as about 1000 times the auto-fluorescence of the respective host cell. Due to the high fluorescence, said cells are very suitable tools in the development, surveillance and/or optimization of production processes, for selection of culture media and process parameters and for the determination of cell growth and the detection, quantification an

Florian Maria Wurm

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Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Production of recombinant protein therapeutics in cultivated mammalian cells

A variant of t-PA (T103N, KHRR 296-299 AAAA) that, by bolus, has increased potency and decreased systemic activation of plasminogen

100-liter transient transfection

Presence and transcription of intracisternal A-particle-related sequences in CHO cells

Chinese Hamster Ovary Cells, Recombinant Protein Production

S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi

Power input correlation to characterize the hydrodynamics of cylindrical orbitally shaken bioreactors

Expression of Herpes simplex virus type 1 glycoprotein C antigens in Escherichia coli

Numerical simulation of orbitally shaken viscous fluids with free surface

Serum-free large-scale transient transfection of CHO cells

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

Effects of methotrexate on transfected DNA stability in mammalian cells

In-Culture Antibody Capture Using Transient CHO Expression Systems

A slow clearing, fibrin-specific, PAI-1 resistant variant of t-PA (T103N, KHRR 296-299 AAAA)

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Generation of recombinant Chinese hamster ovary cell lines by microinjection

GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

Cells, genetically modified with optical marker