High-Level Expression of Recombinant IgG in the Human Cell Line PER.C6 David Jones, Nathalie Kroos, Regina Anema, Bart van Montfort, Andre Vooys, Sven van der Kraats, Esmeralda van der Helm, Shirley Smits, Jan Schouten, Kirsten Brouwer, Fija Lagerwerf, Patrick van Berkel, Dirk-Jan Opstelten, Ton Logtenberg, and Abraham Bout* Crucell, Archimedesweg 4, P.O. Box 2048, 2301 CA Leiden, The Netherlands The number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 × 10 6 cells/mL and produce 300-500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody-producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1-10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high-mannose structures are seen. Introduction Over the past few years, great progress has been made in monoclonal antibody technology (1). There are cur- rently around 10 monoclonal antibodies approved as therapeutics, and a great deal more are in various stages of clinical testing. One reason for this sudden explosion is the development of new technologies which allow the generation of fully human antibodies, including antibody phage display and transgenic mice (which contain in their chromosome key sequences of human antibody genes (2- 4)). Thus, fully human antibodies lacking mouse se- quences can now be made. Antibodies are also glycoproteins, and IgG1 contains a conserved N-glycosylation site at asparagine 297 in the constant region of the molecule. The most complete glycan structure found on an IgG is shown in Figure 1; however, glycans are heterogeneous. In human serum, almost all IgGs contain a biantennary structure with a core fucose. A minor portion (about 10-20%) of these glycans contain a bisecting N-acetylglucosamine, and levels of sialylation have been estimated at 5-20%. In addition, approximately 25% of the structures contain no galactose, 50% contain one galactose residue, and 25% contain two galactose residues (5). The role these glycans play is not fully understood, but the presence of bisecting N-acetylglucosamine appears to increase binding to Fcγ receptors, and galactosylation appears to facilitate in- teraction with complement (6-10). There are also differences in the nature of the glycan structures present on IgGs in different mammalian species (5). This becomes important when antibodies for human therapy are produced in nonhuman cell lines, for reasons of optimal biological activity and potential im- munogenicity (11). It is known that, in some instances, NS0 cells (a mouse myeloma cell line) add a Gal R(1-3) Gal structure to IgG glycans (12); this structure does not exist in humans, and it has been estimated that up to 1% of circulating antibody in humans is directed against this moiety (13). In addition, some cell lines add high- mannose or hybrid structures (14). Production systems for full-length antibodies include expression in mammalian cell lines grown in bioreactors as well as transgenic systems such as plants and larger animals. Important parameters that drive selection of the most optimal expression system for a particular antibody are product quality and quantity, safety issues, time to clinic and market, and economic considerations such as cost of goods and regulatory issues. There is an urgency for further development and optimization of existing technology but also for novel expression technology, * To whom correspondence should be addressed. Phone: +31 (0)71 524 8701. Fax: +31 (0)71 524 8702. E-mail: a.bout@crucell.com. Figure 1. Structure of the most complete glycan found at the conserved N-linked glycosylation site of IgG1. GlcNAc ) N- acetylglucosamine, Man ) mannose, Fuc ) fucose, Gal ) galactose, and Sia ) sialic acid. 163 Biotechnol. Prog. 2003, 19, 163-168 10.1021/bp025574h CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/14/2003