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.