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.

Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein

“…However, a cell line derived from a cabbage looper, Trichoplusia ni BTI-TN-5B1-4 (Hi5), is becoming an increasingly popular choice for infection with BEVS due to the much higher product yield often obtainable with Hi5 compared to Sf-9 (Ikonomou et al 2003;Taticek et al 2001). Sf-9 and Hi5 cell lines, derived from two different organisms, differ markedly from each other in their general behavior and metabolism.…”

An actively mixed mini‐bioreactor for protein production from suspended animal cells

“…Previous work (Taticek et al, 2001) has shown that 100 rpm was an optimal agitation speed to obtain high specific growth rate of Sf-21 cultured in 50 mL spinner flasks and reported a similar specific growth rate of 0.034 h À1 . However, cells grown in mini-bioreactors reach a higher total density of 5.3 AE 0.9 Â 10 6 cell/mL at the stationary phase than cells grown in spinner flasks with the maximum cell density of 3.4 AE 0.4 Â 10 6 cell/mL.…”

Microbial Expression Systems and Manufacturing from a Market and Economic Perspective

“…As for other cell lines used for production, insect cell lines need to be "immortalized" or rendered permanent. (Taticek et al, 2001) expressing Escherichia coli beta-galactosidase or human secreted alkaline phosphatase. The production of both enzymes varied as a function of inoculum size, media, culture conditions etc.…”