title: Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture.
abstract: Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-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 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 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 beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-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 beta-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 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-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 beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-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.
authors.affiliation: School of Chemical Engineering, Cornell University, Ithaca, New York 14853-5201, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Taticek
authors.forename: C
authors.initials: C
authors.lastname: Choi
authors.forename: S E
authors.initials: SE
authors.lastname: Phan
authors.forename: L A
authors.initials: LA
authors.lastname: Palomares
authors.forename: M L
authors.initials: ML
authors.lastname: Shuler
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D016895:Culture Media, Serum-Free; D007313:Insecta / Q000166:cytology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase