Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Abstract

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development

“…The growth kinetics of the SMC-1 line achieved in a shake flask and an airlift bioreactor showed the absence of a lag phase, which could be due to different factors such as the physiological state of the inoculum at the time of initiating the kinetics. Subsequently, exponential growth continued from day 10 to 12; this is comparable to earlier studies with Daucus carota , which correlated to 10 and 15 days 24 . In this exponential stage, the cells present a higher capacity for growth and division because of the immediate availability of nutrients.…”

“…The pattern in the consumption of glucose and fructose was similar; however, a preferential consumption of glucose with respect to fructose was observed. This behavior has been reported in other suspension cell cultures 24 . The variation in pH medium in the shake flask and bioreactor culture could be related to the uptake and utilization of the nitrogen source in the culture medium by the cells.…”

“…In this exponential stage, the cells present a higher capacity for growth and division because of the immediate availability of nutrients. The growth rate and doubling time recorded in the airlift bioreactor was shorter than in the shake flask from µ= 0.58/d to 0.3/d and td from 1.3 to 2.3 days; these outcomes are comparable with other suspension cell cultures in which td values range from 1.1 to 4.6 days and growth rate (µ) values range from 0.074 to 0.259/d 24 . These differences between shake flask and bioreactor were related to the inoculum size (10% and 5%, respectively) and mainly to the operating conditions in the bioreactor such as the direct air spray, which promotes the circulation of nutrients in the culture.…”

“…Growth kinetics was initiated with 10 % (v/v) of cell suspension cultures of the SMC-1 line in Erlenmeyer flasks with 100 mL of B5 medium and maintained under orbital shaking at 120 rpm under continuous light condition (50 µmol/m 2 /s) at 25 ± 2 °C for 30 days. Kinetic parameters such as fresh and dry weight (FW and DW), percentage cell viability, pH, and carbohydrate consumption were determined according to previous reported techniques [24].…”

“…The airlift bioreactor was operated at 0.5 vvm for 26 days and maintained at 25 ± 2 °C under continuous light (50 µmol/m 2 /s). The kinetic parameters were calculated as previously reported [24].…”