3.13 Membrane testing
4.1.1 Virus replication and scale-up into bioreactors
First, a working virus seed was generated from egg-derived YFV-17D virus material. The lyophi-lized virus was reconstituted in PBS and used to infect 90 % confluent Vero cells in four tissue culture flasks (each 175 cm² surface). The cell supernatant was collected 96 hpi and stored at
−80 °C. Pooled virus harvests had a titer of 2.1×108 PFU/mL. The virus seed was used for all in-fection studies of Vero cells in tissue culture flask and microcarrier-based bioreactor cultivations.
Cells grew in serum-containing Z-Medium and experiments were performed as described in sec-tions 3.5 and 3.7, respectively.
Tissue culture flask cultivations Vero cells were cultivated in tissue culture flasks (25 cm²) until 90 % confluency was reached (based on visual inspection of surface area). Cells were then infected with an MOI 2×10-21. Infected cells continued growing and maintained high viabilities for 5 days
1 Note: Calculated MOI and measured MOI at time point of infection (toi) can vary by a factor of 5 to 10 due to virus inactivation after seed virus thawing. As a means for comparability, hereinafter MOIs are referred to as MOIs
4.1 Yellow fever virus production with adherent Vero cells
61 (based on trypan blue exclusion). Then, cells started to detach, and viabilities strongly decreased.
In parallel, the virus titer in the supernatant was measured. During the first 24 hpi, YFV titers dropped slightly and then increased to peaking titers of 1.9×107 PFU/mL after 96 hpi (Fig-ure 4.1 A). In total, a maximum cell-specific virus yield of 11.9 PFU/cell was obtained (Table 4.1).
Microcarrier cultivations Static cultivations performed in tissue culture flasks and other cultiva-tion systems have limited opcultiva-tions for full process monitoring and control. Thus, a scalable produc-tion process was evaluated using Cytodex-1 microcarriers in quasi-suspension maintained in ner flasks and in controlled bioreactor systems (Chapter 3.5). First, cells were inoculated to a spin-ner flask at a seeding concentration of 29 cells/MC in Z-Medium (ratio chosen based on fast adhe-sion kinetics described by Bock for Vero cells [215]). When cells reached a cell concentration of 1×106 cells/mL, cells were infected with YFV at MOI 4×10-2. Cells continued growing until full confluency. Finally, a virus titer of 8×106 PFU/mL was achieved resulting in a cell-specific virus yield of 3.6 PFU/cell (Figure 4.1 B, Table 4.1). During the cultivation, the pH value decreased con-stantly to 6.9 known to be suboptimal for YFV production (not shown here). To maintain pH values of 7.2, the process was transferred to a 1 L stirred-tank bioreactor vessel. With the typical impeller configuration for suspension cells, it required high stirrer speeds to maintain confluent microcarri-ers in quasi-suspension. However, the resulting, high shear stress on cells and carrimicrocarri-ers led to poor cell growth and microcarrier damage. Hence, the stirrer unit was adjusted by mounting two segment impellers in bidirectional axial flow direction. As a result, confluent microcarriers were kept in quasi-suspension and intact at stirrer speeds of only 60 rpm. Cells grew at controlled pH and pO2
conditions to a final concentration of 1.7×106 cells/mL (Figure 4.1 C). YFV titers reached 2×107 PFU/mL resulting in a cell-specific titer of 10.8 PFU/cell. Based on the microcarrier surface area (as specified by the manufacturer), maximum surface-specific cell concentrations (cell satura-tion density) decreased from 7.2×105 cells/cm² in tissue culture flasks to 1.3×105 cells/cm² in stirred-tank bioreactors indicating lower cell growth on available surface, either due to morpholog-ical changes (i.e. reduced height and larger area to keep similar cell volume at higher shear condi-tions) or due to simple overestimation of available surface area as stated by the manufacturer (Ta-ble 4.1).
0 48 96 144 192 240 0.0
0.5 1.0 1.5 2.0 2.5
1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09
0.0 0.5 1.0 1.5 2.0 2.5
1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09
Cell concentration (´106 cells/mL)
Cultivation time (h)
0 48 96 144
0.0 0.5 1.0 1.5 2.0 2.5
1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09
Cultivation time (h)
0 48 96 144 192
0.0 0.5 1.0 1.5 2.0 2.5
103 104 105 106 107 108 109
Cultivation time (h)
YFV titer (PFU/mL)
Figure 4.1 │ Yellow fever virus production in adherent Vero cells at different scales. Viable Vero cell concentration (blue square) and infectious YFV titer (red triangle) in (A) tissue culture flasks with a surface of 25 cm², (B) in spinner flasks with 3 g/L Cytodex-1 microcarriers, and (C) in controlled bioreactors with 3 g/L Cytodex-1 microcarriers. Dotted vertical line indicates time point of infection.
A B C
Chapter 4 Results and Discussion
Table 4.1 │ Process results from yellow fever virus production in adherent Vero cells at different scales.
Scale Viabl. Cell conc.
(×106 cells/mL)
Surface-spec. VCC (×105 cells/cm²)
Maximum YFV titer (×107 PFU/mL)
Cell-spec. virus yield (PFU/cell) *
T-flask a 1.8 7.2 1.9 11.9
Spinner with MC b 2.2 1.7 0.8 03.6
Bioreactor with MC b 1.7 1.3 1.8 10.8
VCC = viable cell concentration; surface-specific viable cell concentration is calculated with surface areas as provided by manufacturer: a 25 cm² surface area; b 3 g/L microcarrier concentration; * calculated according to maximum cell concentra-tion until time point of highest virus titer (see Equaconcentra-tion 18).
Bead-to-bead transfer Major constraints of microcarrier-based cultivations emerge when me-chanical or enzymatic treatment is required to harvest confluent cells for the transfer into larger scales. Hence, a direct bead-to-bead transfer was investigated for Vero cells in Z-Medium. Previous inoculation experiments with 29 cells/MC revealed a ratio to colonize sufficient all microcarriers.
After 72 h, empty microcarriers were added to the near confluent ones (103±38 cells/MC). To fa-cilitate direct bead-to-bead contact, agitation was periodically stopped, i.e., 5 min agitation and 25 min static incubation. The cell spreading was monitored for the next 96 h (Figure 4.2). Within 2 h, first cells were already found on newly added microcarriers and 18 h later, microcarriers at-tached to each other by formation of cell bridging in quasi-suspension. After 96 h, all microcarriers became near to confluency and first cells started to detach.
Figure 4.2 │ Evaluation of bead-to-bead transfer for the scale-up of Vero cells on Cytodex-1 micro-carriers. Vero cells grew on microcarriers (3 g/L) in Z-Medium and reached near confluency 72 h after inoculation. Empty microcarriers (1.5 g/L) were added and agitation was periodically stopped (time point 0 h). Cells started to spread to newly added microcarriers until all beads showed full confluency (96 h).
Scale bar indicates 150 µm.
0 h 2 h
18 h 96 h
4.1 Yellow fever virus production with adherent Vero cells
Discussion First infection experiments confirmed that Vero cells are permissive for YFV. Shortly after infection, virus titers in the supernatant decreased, presumably due to virus adsorption to the cell and entry. After one day, extracellular titers increased indicating a slow virus replication of about 24 h. Finally, infectious titers peaked around 3-5 days post infection and decreased after-wards. Thereby, cell viabilities remained high over extended periods after infection and eventually decreased. This is consistent with general observations of YFV, which is considered to be a (slow) lytic virus. Flaviviruses are known to trigger cell survival as well as cell death via many signaling pathways leading to apoptosis, necrosis and autophagy. Unlike lytic viruses, flaviviruses can lose their ability to induce apoptosis in certain cells by inhibiting the type I interferon (IFN-I) signaling pathway [216, 217]. This may play only a minor role here, as Vero cells are known to be resistant to antiproliferative effects of IFN [218]. In the case of other cells, this strategy may be beneficial from the evolutionary perspective of the virus as infected cells are temporarily spared, which allows longer time periods of replication and enhanced viral yields. Overall, in-vitro experiments with Vero cells resulted in only low infectious virus titers of about 10 infectious virions per cell (PFU/cell) in agreement with reports from other workgroups (RKI Berlin, Prof. Niedrig; UFRJ, Prof. Tanuri) and industry (Valneva, Dr. Léon). The controlled bioreactor set-up enabled stable pH control at 7.2 known to be optimal for ZIKV replication [219]. In contrast, headspace aeration, as present for spinner flasks, was not sufficient to maintain preferred pH values in Z-Medium, poten-tially inactivating YFV titers at pH 6.8 and explaining the low infectious titers [220].
During the scale-up from tissue culture flasks to microcarrier cultivations, surface-specific cell concentrations decreased strongly by a factor of 5. This implies that nominal surface areas (as given by the manufacturer) may either not match to real surface available for cell growth or that morpho-logical changes reduced cell saturation densities. In general, herein reached cell concentrations of approximately 6×105 cells/gMC and cell concentrations are in good agreement with published data [221, 222]. Interestingly, cell concentrations on microcarrier beads reached a plateau at about 24 h after inoculation. This is typically observed during the so-called ‘cell adhesion phase’ between 0-24 h [205]. Attached cells quickly increase in cell diameter before cells start dividing into daugh-ter cells with smaller diamedaugh-ters [223]. This finding can be confirmed by online cell volume data and is further elaborated in section 4.1.2. The bead-to-bead transfer is an attractive solution for the scale-up process [224]. Fortunately, Vero cells in Z-Medium successfully transferred to newly added beads after intermittent agitation. This enabled a direct cell expansion to the next process scale. However, it also results in very unsynchronized cell stages, where the parental cell is typically surface-limited and may undergo cell apoptosis, while daughter cells on empty microcarriers still divide. The function of formerly colonized microcarriers and resulting available surface area re-mains questionable. This may also explain that most manufacturers harvest the cell seed inoculum from static multilayer cultivations.
Previous attempts for inactivated YFV-17D and 17DD vaccine production with Vero cells on microcarrier beads were reported by Xcellerex (US) and Fiocruz (Brazil), respectively [8, 9]. In both studies, virus titers exceeded 108 PFU/mL. Certain details on process conditions revealed an increase in infectious titers by the use of serum-free virus production media and medium exchanges during YFV production phases. Therefore, similar strategies, i.e. biphasic process for optimum cell growth and virus production, serum-free media screening, medium exchange before infection, tem-perature decrease, optimized MOI and time point of infection (toi) optimization, can be considered equally. In addition, further process intensification strategies can be envisaged to increase cell
Chapter 4 Results and Discussion
numbers by higher microcarrier concentrations (limited by bead collisions [7]) or the use of fixed-bed bioreactors in combination with perfusion modules [7]. However, adherent cells will always have limitations in scale-up eventually reducing the interest to change current egg-based production processes. Alternatively, suspension cells enable the establishment of various process intensifica-tion strategies with an easy transferability into manufacturing scale. Hence, the future focus should be on suspension cells in comparison to the Vero cell-based production process as reference stand-ard.