Gene transfer and gene amplification in mammalian cells Florian M. Wurm and Martin Jordan 1. Introduction on the origin of Chinese hamster ovary cells for recombinant protein production Mammalian genomes have exceptional plasticity. They can exhibit short-term plasticity as evidenced by the fact that some DNA sequences, whether endogenous or introduced by artificial gene transfer, can be induced and/or selected for gene amplification to high copy numbers. In addition, mammalian genomes have an apparently unlimited capacity to take up foreign DNA, the hallmark of this being the presence of retroviral and bacterial DNA sequences that have become an integral part of these genomes. The short-term plasticity and ability to take up DNA makes cell lines useful for the expression and production of recombinant proteins of scientific and pharmaceutical value. Foreign genes can be easily integrated into mammalian cells cultivated in vitro and, through selection, populations of cells of clonal origin can be identified that show an increased copy number of the inserted genes. These cell lines express the required genes of interest at elevated levels. This approach has been used widely in both industry and academia, particularly with two immortalized cell lines, Chinese hamster ovary (CHO) cells and a mouse myeloma cell line, NS0. We will primarily concentrate on the dhfr/CHO system, since it is the most commonly used system. In general, most of the observations made with this system apply to other immortalized cell lines and thus should give insights into other types of gene transfer and amplification systems in mammalian cells. CHO cells remain the most popular mammalian host for recombinant protein production since the mid 1980s, when the first product, human recombinant tissuetype plasminogen activator Activase_ (rTPA), received regulatory approval for marketing. These cells have also been a preferred choice for studies on gene transfer and gene amplification in mammalian cells. Some of the studies were driven by questions raised by regulatory authorities controlling the clinical evaluation and marketing of pharmaceutical products. Other motivations were the need to maximize S.C. Makrides (Ed.) Gene Transfer and Expression in Mammalian Cells _ 2003 Elsevier Science B.V. All rights reserved

gene expression from transfected and amplified plasmid sequences and to establish recombinant cell lines with long-term stability with regard to the genome and to protein productivity. The popularity of CHO cells in recombinant protein production is based on both practical and virological safety reasons. Discussions on virus safety with mammalian production host systems started a half century ago when the first biological products, i.e., vaccines and natural interferons, were developed using primary and transformed mammalian cells. It is an essential goal both for the manufacturers of recombinant protein pharmaceuticals and for regulatory agencies to minimize any potential risks associated with the use of immortalized recombinant mammalian cell hosts. CHO cells were established for the study of somatic cell genetics in 1957 by Puck [for review see [1]]. These cells are well characterized with respect to karyotype, chromosome structure, gene mapping and culture conditions. Upon the emergence of DNA technology one particular derivative of the original CHO cells (see below) was used in a number of pioneering gene transfer experiments [2,3]. It was possible to sub-cultivate these cells after the identification of genetically altered candidate clones producing the protein(s) of interest under increasingly selective conditions. This resulted in the generation of more productive amplified subclones. Another key feature for industrial scale production was the finding that CHO cells can be easily adapted to grow in suspension. In the mid 1980s R. Arathoon, A. Lubiniecki, G. Polastri and others at Genentech Inc. in San Francisco developed a 10,000-liter production process for rTPA, using deep tank technology with suspensionadapted

CHO cells [4]. CHO cells are considered to be a safe host for the production of therapeutic proteins, which will be administered parenterally to human patients. Human pathogenic viruses like Polio, Herpes, Hepatitis B, HIV, Measles, Adenoviruses, Rubella and Influenza do not replicate in these cells. Thus, the risk of a viral adventitious agent being involuntarily carried along with the product of interest is low. Wiebe et al. tested a total of 44 human pathogenic viruses for replication in CHO cells and found that only seven of these (Reo 1,2,3, Mumps, and Parainfluenza 1,2,3) were able to infect these cells [5]. These safety considerations were discussed extensively among scientists from both regulatory agencies and the bio-pharmaceutical sector. Today one can only speculate whether an immortal human cell substrate with replicative capacity for a larger number of human viruses would have received approval for the production and marketing of a protein therapeutic. Only very recently (2002) was the marketing approval given for a protein pharmaceutical produced in the immortalized human cell line HEK-293 (Activated Protein C, requiring C-terminal carboxylation, E. Lilly). Due to complex processing and post-translational modifications, many therapeutic proteins must be produced in mammalian cells. At present (2003), at least 50 recombinant proteins made in CHO cells have received approval for marketing. It is difficult to assess the exact number of CHO-based product candidates that are in pre- and clinical evaluation, but it is with certainty above 200.