LEGENDS OF FIGURES
Figure 1: Generation of cell pools named 1 and 2, enriched by means of 1 or 2 rounds of FACS for YF-VLP high-producers, respectively. (A) Passage history showing viable cell density (VCD) and viability of mock transfection control, of original stable cell pool HEK293-YF and of enriched cell pools 1 and 2. The first round of cell sorting was performed 40 days after transfection (vertical red dashed line) and the second round 98 days after transfection (vertical blue dashed line). (B) Comparison of VLP production by cell pools 1 and 2 generated by FACS-enrichment. Dengue envelope protein (Prospec, Israel) was used as positive control and pan-flavivirus monoclonal antibody 4G2 was used as primary antibody.
Figure 2: Cell growth and glucose and lactate concentrations along time for cell pools 1 and 2 cultivated in shaken vented tubes in batch and pseudoperfusion (PP) modes. (A) Viable cell density (VCD) and viability of cell pools 1 and 2 grown in batch mode. (B) VCD and viability for cell pools 1 and 2 cultivated in pseudoperfusion mode. (C) Glucose (Glc) and lactate (Lac) concentrations along batch cultures of cell pools 1 and 2. (D) Glucose concentration and daily medium exchange rate applied during PP experiment with cell pools 1 and 2. Feeding strategy started on day 4 and samples were taken before and after feed addition. (E) Yellow fever VLP production for cell pools 1 and 2 when cultivated in batch and pseudoperfusion modes.
Figure 3: Production of YF VLPs by cell pool 1 cultivated in perfusion mode using ATF-2 as cell retention device. (A) Viable cell density (VCD), online biomass measurements and viability during perfusion cultivation. (B) Glucose and lactate concentrations and rates of medium addition (D_feed) and harvest (D_harvest) during perfusion cultivation. Due to membrane fouling, D_feed was higher than D_harvest from day 10 on. (C) VLP concentration inside the bioreactor and in the harvested perfusate.
Figure 4: Production of YF VLPs by cell pool 2 cultivated in perfusion mode using the inclined lamella settler CS-10 as cell retention device. (A) Viable cell density (VCD) and viability during perfusion cultivation. (B) Glucose and lactate concentrations and medium exchange rate during perfusion cultivation, given as volume of fresh medium per culture volume per day. (C) VLP concentration inside the bioreactor and in the harvested perfusate.
Figure 5: VLP purification from cell culture supernatant. (A) Typical chromatogram for steric exclusion chromatography (SXC): the solid line represents the absorbance at 280 nm and the dashed line the PEG 6000 concentration. (B) Immunoblot of the fractions collected during the chromatography using the pan-flavivirus monoclonal antibody 4G2 as primary antibody. (C) Silver-stained SDS-PAGE. Lane 1: molecular mass marker. Lane 2: clarified supernatant. Lane 3: purified VLP (SXC eluate). (D) Western blot analysis using 4G2 as primary antibody. Lane 1: molecular mass marker. Lane 2: clarified supernatant. Lane 3: purified VLP (SXC eluate). Lane 4: purified VLP after ultra and diafiltration for concentration for PEG removal. (E) SE-HPLC chromatograms using the TSKgel G3000SWXL column for cell culture supernatant and SXC-purified VLP samples.
Figure 6: Characterization of SXC-purified yellow fever virus-like particles. (A) Zoomed panel of images acquired by negative-staining transmission electron microscopy (scale bar: 30 nm). (B) Line plots showing size exclusion chromatograms using a Superose 6 10/300 column.