Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa,
Faculdade de Ciências e TecnologiaThe present work focuses the optimization of the production of human Leukemia
Inhibitory Factor (hLIF) by human embryonic kidney 293 (HEK293) - EBNA1 cell line
cultured as suspension aggregates in spinner flask. The effects of initial cell density and feeding-regime in cellular growth and productivity were evaluated.
A first phase occurs until the end of exponential growth in medium containing
serum, being followed by puromycin selection of cells containing hLIF plasmid. A
second phase, the production phase, is developed under serum-free conditions. Three
initial cell densities (2´104 cells.mL-1, 2´105 cells.mL-1 and 2´106 cells.mL-1) were
tested and the effect on maximum cell density and cell aggregate size distribution was evaluated. The inoculum of 2´105 cells.mL-1 led to final higher cellular densities around 7´106 cells.mL-1 and homogeneous aggregates around 278 μm were observed. The effect of the feeding-regime was then studied for the production of recombinant hLIF with an initial cell density of 2´105 cells.mL-1 by performing metabolite analysis Metabolite analysis revealed the occurrence of glucose and glutamine starvation upon 9 and 7 days, respectively, in 25% FR. Therefore, glutamine acted as an alternative carbon, energy and aminoacid source in HEK293-EBNA1 growth. Observed lactate levels were bellow the inhibition limit concentrations (30 mM 1).HEK293-EBNA1 did not seem to be affected by the produced levels of ammonia.
In conclusion, the 25% feeding regime at 2´105 cells.mL-1 initial cell density
lead to the higher viable cell densities, either along culture time at growth and at
production phase, with aggregates in suspension below necrotic diameters. This
experiment conditions also allowed achieving the higher LIF volumetric productivity,
125 ± 2.2 ng.mL-1
