Boehringer Ingelheim Pharma Gmbh & Co Kg

Juergen Fieder

Ralf Otto

Barbara Enenkel

Klaus Bergemann

Thomas Krieg

Kerstin Sautter

Hitto Kaufmann

Lore Florin

Stefanos Grammatikos

Rebecca Bischoff

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 present invention refers to the area of cell culture technology and relates to methods for multiplying/cloning cells, preferably cell lines, which are important to the production of biopharmaceuticals. The invention further relates to methods for manufacturing proteins and to the use of cells extracted and multiplied through single cell sorting and to media compositions that enable a multiplication of single cells. Through the use of albumin-producing, preferably HSA-producing, cells as feeder cells for the conditioning of medium or as host cells, the recloning efficiency and thereby the quantity of clones obtained can be significantly increased. A combination of these approaches is also possible. Through the use of albumin-producing, preferably HSA-producing, cells, an increase in the recloning efficiency can be achieved in serum-free and/or insulin-free medium as well, and in different cell types.

Use of HSA-producing 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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Unterstadion, Germany

Juergen Fieder