3.5 scFv-Fc production by CHO/GEV_scFv-Fc cells in
semi-continuous culture at high density
Next, we attempted continuous production under high-cell-density culture
conditions with 4-OHT induction using a bioreactor tube (Figure 5). As
described above, our results reveal that antibody production could be
induced without significantly impacting cell proliferation under the 0.1
µM 4-OHT condition. We also found that the maximum concentration and
specific productivity of scFv-Fc could be achieved within 3 to 4 days
after adding 4-OHT. Following pre-culture for cell proliferation without
4-OHT, we initiated suspension cultures with high cell density by
switching to 4-OHT-containing medium. Semi-continuous culture was
carried out for more than 2 weeks to evaluate the stability of induced
scFv-Fc production under the 0.1 µM 4-OHT condition. When seeded at a
density of 1.0 × 107 cells/mL, cell proliferation was
observed (Figure 5A) but a decreased level of cell viability was
maintained around 80% (Figure 5B). scFv-Fc concentrations demonstrate
stable production in the medium at an average of 0.44 ± 0.05 g/L over
the duration of culture (Figure 5C), with a specific productivity of 37
± 4 pg cell–1 day–1 (Figure 5D).
In contrast, with a seeding density of 0.5 × 107cells/mL, nearly 100% viability was maintained throughout the culture
period (Figure 5B) and scFv-Fc production at a concentration of 0.31 ±
0.03 g/L (Figure 5C) and specific productivity of 57 ± 5 pg
cell–1 day–1 (Figure 5D) was
stably achieved. Analysis of metabolites in the spent medium indicate
glucose concentrations of 4.3 ± 0.9 mM and 2.0 ± 0.7 mM for seeding
densities of 0.5 × 107 cells/mL and 1.0 ×
107 cells/mL, respectively (Figure 5E). Lactate
concentrations were measured to be 34 ± 6 mM (0.5 ×
107 cells/mL) and 66 ± 7 mM (1.0 ×
107 cells/mL) (Figure 5F). Conversion rates from
glucose consumption to lactate production were estimated to be 52% (0.5
× 107 cells/mL) and 100% (1.0 × 107cells/mL).
scFv-Fc protein produced in the medium of semi-continuous cultures was
analyzed using SDS-PAGE. The results show that bands of the expected
molecular weight were detected under both reducing (Figure 6A and 6C)
and non-reducing (Figure 6B and 6D) conditions, indicating that the
protein was produced in an intact form (Figure 6).
Taken together, these results demonstrate that our estrogen
receptor-based inducible expression system can be applied for
high-cell-density semi-continuous culture to stably produce recombinant
proteins for over 2 weeks.