Boehringer Ingelheim Pharma Gmbh & Co Kg

Ralf Otto

Juergen Fieder

Barbara Enenkel

Klaus Bergemann

Kerstin Sautter

Thomas Krieg

Hitto Kaufmann

Stefanos Grammatikos

Jurgen Fieder

Jürgen Fieder

A new method for selecting clones and recloning mammalian cells which are of importance for the production of biopharmaceuticals, preferably hamster or mouse myeloma cells, with a high degree of automation and throughput. The invention relates to methods of depositing and replicating single cell clones of the cells in question. The invention also relates to methods of preparing proteins using cells which have been obtained and replicated by single cell deposition as well as compositions which allow the replication of single cells.

Methods of producing proteins in Chinese hamster ovary (CHO) cells

The invention relates to modified neomycin phosphotransferase genes and their use in a selection method for high-producing recombinant cells. The invention further relates to expression vectors which contain a modified neomycin phosphotransferase gene and a gene of interest functionally linked to a heterologous promoter and a method of preparing heterologous gene products using these expression vectors.

Neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product

Method for recloning Chinese hamster ovary (CHO) cells

Production of biopharmaceuticals from CHO cells requires generation of master-, working- and post-production cell banks of high quality, partly under GMP conditions. An optimal cryopreservation strategy is needed for each new production cell line, particularly with regard to the desire to establish production processes that are completely devoid of serum or even any animal components and to ensure robust thaw performance for reliable production. Here we describe a novel strategy employing flow cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of CHO cell banks. Our data show that this method enables evaluation of a cryopreservation procedure just 6 h after thaw.

Use of flow-cytrometric analysis to optimize cell banking strategies for CHO cells

An expression vector for eukaryotic cells comprising a gene which codes for a protein of interest, functionally linked to a hamster-ubiquitin/S27-promoter and a gene which codes for a fluorescent protein. Preferably the expression vector also contains an amplifiable selectable marker gene. The invention also describes host cells, preferably mammalian cells, which have been transfected with the expression vector, processes for producing heterologous gene products and a method of selecting high-producing cells.

Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such products

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Oggelshausen, Germany

Ralf Otto