Comparison of Growth and Recombinant Protein Expression in Two Different Insect Cell Lines in Attached and Suspension Culture R. A. Taticek, ²,| C. Choi, ² S-E. Phan, ² L. A. Palomares, ‡ and M. L. Shuler* ,² School of Chemical Engineering, Cornell University, Ithaca, New York 14853-5201, and Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca, Mor. 62271, Mexico Culture conditions required for obtaining maximum recombinant protein concentra- tions from two cell lines, Spodoptera frugiperda (IPLΒ-Sf21-AE) and Trichoplusia ni (Tn 5Β-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Β-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 × 10 5 cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for -galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Β-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of -galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM- FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Β-1-4 cells produced 2.6-4.4 and 2.7-3 times more -galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900- II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX- CELL 400 performed better than the other two media, as it increased the -galac- tosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of -galactosidase by Tn 5Β-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production. Introduction Insect cells are an attractive host for recombinant protein expression via infection with a genetically modi- fied baculovirus as this system allows for high levels of production of proteins that are functionally similar to the native protein (1). A wide range of procaryotic, eucaryotic, and viral genes have been expressed using this system (2). For the insect cell-baculovirus expression system to be readily adopted as a method for large-scale production, a number of issues must be addressed. Larger scale efforts have focused on suspension culture, since most insect cell lines can be manipulated to grow either in suspension or attached. An attached cell reactor would be considered if a compelling reason exists, such as vastly superior production or enhanced product quality due to more complete post-translational modification. Attached cultures would be also useful for perfusion reactors. It is unclear from the literature as to whether attached or suspension cells express more protein. Lanford reports 3-5 times higher specific production in adhered cells than in suspension culture (3), while Maiorella et al. report a 3-fold improvement in M-CSF production going from attached to suspension culture (4). Murhammer and Goochee (5) and Sugiura and Amann (6) obtained similar recombinant protein concentrations in attached and suspension systems. The cost, lot-to-lot variability, and downstream pro- cessing difficulties associated with the use of serum- supplemented media or high protein containing serum- free media (SFM) has led to the development of a number of low-protein serum-free media for use with insect cells (4, 7, 8, 9, 10). These SFM generally consist of a basal medium (as IPL-41) supplemented with yeastolate and a lipid emulsion (containing cholesterol, a-tocopherol acetate, cod liver oil, Tween 80, and pluronic F-68) among other components. Some of these components are unde- fined and subjected to lot-to-lot variations. Moreover, * Correponding author. Tel: 607/255-7577. Fax: 607/255-9166. E-mail: mls@cheme.cornell.edu. ² Cornell University. ‡ Universidad Nacional Autonoma de Mexico. | Present Address: Genentech, Cell Culture Fermentation R&D, 460 Point San Bruno Boulevard, South San Francisco, California 94080. 676 Biotechnol. Prog. 2001, 17, 676-684 10.1021/bp010061g CCC: $20.00 © 2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/29/2001