Growth of clone P/cSIN/92 in different media with or without nutrient supplement:
The growth of clone P/cSIN/92 was evaluated in six commercial media with or without supplement of the two commercial feeds, CB5 and HEK FS. SFM4transfx 293 was used as a basal medium in previous studies and included in this experiment as a reference. The cell growth in FreeStyle F17 slowed down after one passage and stopped after three passages. Experimental results from other five media and two feeds are shown in Figs. 2 and 3, respectively. The data in Fig. 2 revealed that the clone only grew to a maximum viable cell density of 2.7 × 106 cells/mL and 3.7 × 106 cells/mL in batch culture, respectively, using BalanCD HEK293 or SFM4transfx 293. A significant higher cell density, 5.7 × 106 cells/mL, was achieved when clone P/cSIN/92 was cultivated in HyCell™ TransFx-H, and a cell doubling time was 27 h during the exponential growth phase. Feeding the HyCell™ TransFx-H culture with 3% and 5% CB5 feed, respectively, on days 5 and 6 increased the maximum viable cell density to 8 × 106 cells/mL.
The cell growth was promising in HEK GM and HEK TF (both media from Xell AG), respectively, reaching 6.5 and 8.1 × 106 viable cells/mL in batch cultures (Fig. 3). Feeding the HEK TF culture with HEK FS feed increased the maximum viable cell density from 8.1 × 106 cells/mL to 8.8 × 106 cells/mL. The higher cell density (> 8 × 106 cells/mL) achieved in the cultures using HyCell™ TransFx-H and CB5 or HEK TF and HEK FS feed provides opportunities to explore the induction of clone P/cSIN/92 culture at high cell density (for example 5 × 106 cells/mL) for possible higher titer production of LVs. Consequentially, HyCell™ TransFx-H and CB5, HEK TF and HEK FS feed were selected for further experiments aimed to improve LV productivity.
Production of LV in clone P/cSIN/92 cultures grown with HyCell TransFx-H or HEK TF without feeding before or after induction
Batch culture was first conducted to evaluate LV productivity in cultures grown in the two identified top performance media, HyCell TransFx-H or HEK TF, and induced at a cell density of 1 × 106 cells/mL. In parallel, a culture grown in SFM4transfx 293 to 1 × 106 cells/mL and induced without medium exchange was conducted as a reference. Figure 4 showed the LV productivity was very poor, at 9.75 × 105 TU/mL, in the culture grown with HEK TF and induced without medium exchange. This titer was lower than the 9.6 × 106 TU/mL achieved in the reference with SFM4transfx 293. A medium exchange by resuspending the cells grown in HEK TF to fresh HEK TF or HyCell TransFx-H medium before induction restored the LV production to1.2 × 107 and 1.4 × 107 TU/mL, respectively. In contrast, the LV production was much better in the culture grown with HyCell TransFx-H, and was at 1.57 × 107 TU/mL in the culture induced without medium exchange. The titer was further improved and reached 2.17 × 107 TU/mL in the culture when a medium exchange with fresh HyCell TransFx-H was carried out. This result may suggest that nutrients required in the phase of LV production were sub-optimal in the HEK TF medium, and quality of the cells grown in HEK TF was also sub-optimal for promoting the virus production, even though HEK TF supported the clone growing to high cell density. In comparison, HyCell TransFx-H is better formulated to support the LV productivity of clone P/cSIN/92 and was, therefore, used in further experiments aiming to improve LV production.
Batch culture was then explored to examine a possibility of improving LV productivity through induction of batch culture at different densities. Data in Fig. 5 reveal that the volumetric LV titer was improved from 1.6 × 107 to 2.5 × 107 TU/mL (or up to 53% increase) when the cell density at induction was increased from 1.3 to 3.1 × 106 cells/mL. With further increase of cell density at induction to 3.8 × 106 cells/mL, the virus productivity dropped to 1.5 × 107 TU/mL or 92% of that obtained in the culture induced at 1.3 × 106 cells/mL (Fig. 5). When converting the above volumetric productivity into the cell specific vector productivity, which is calculated by dividing the volumetric productivity by the cell density at induction, it shows that the cell specific vector productivity declined from 12.3 to 3.9 TU/mL when the cell density at induced increased from 1.3 × 106 to 3.8 × 106 cells/mL.
One set of experiment was conducted to examine whether the declined cell-specific vector productivity was due to change of the “quality” of clone P/cSIN/92 grown to high cell density. In this experiment, cultures grown to different cell densities were centrifuged, respectively. Cell pellet was then resuspended to fresh HyCell TransFx-H medium at a final density of 1 × 106 cells/mL before induction to ensure that all cells from different density cultures were in the same culture environment (such as available nutrients) during the vector production phase. Data in Table 1 reveal that the cell-specific vector productivity was about 17 TU/cell for the cells taken from the three cultures grown to different densities, suggesting the cells prior to induction were in the same state (or “quality”) in terms of supporting the virus production. The 25.7 TU/cell for the cells from the culture at a density of 2.74 × 106 cells/mL was significantly higher, which could be due to deviation of experimental conditions.
Production of LV in clone P/cSIN/92 culture grown HyCell TransFx-H in high cell density culture with nutrient supplement and/or medium exchange before induction
Several sets of experiments were dedicated to investigate if the LV productivity could be significantly increased by inducing the culture at high cell density (such as 5 × 106 cells/mL) and through feeding the culture prior to and/or post the induction. The tested nutrients (feeds) included CB5 from 1 to 5%, yeast extract (Biospringer) at 2–4 g/L, insulin (10–100 µg/mL, ThermoFisher Scientific) or a combination of different nutrients. Results from these sets of experiments revealed that, when compared to the culture induced at 1 × 106 cells/mL, improvement in the LV volumetric productivity was marginal except 2.2-fold increase achieved in the culture induced at 5 × 106 cells/mL with a complete medium exchange before the induction (Table 2). The LV titer actually declined in the culture only fed with 3% or 5% CB. Data from additional test on the effect of CB5 supplement (not shown) suggest that CB5 feed might interfere with the induction and/or vector production.
Spent media from non-induced or induced cultures were analyzed for concentration of residual nutrients and inhibitory metabolites, typically amino acids, glucose, lactate and ammonia. Figure 6 depicts the residual concentration of amino acids, glucose, lactate and ammonia at inoculation, induction and 72 hpi in an exemplary culture fed with CB5 and induced at 5 × 106 cells/mL without medium exchange. These results show that amino acids and glucose were not depleted while the concentration of lactate and ammonia was within a normal range, suggesting limiting nutrient(s) or inhibitory metabolite(s) were not the above monitored components. Feeding induced culture with individual component might not be an effective and fast approach to improve or optimize nutrient supply and to increase the virus production, considering complexity of culture media consisting of up to 100 components.
Synergetic effect of mixing culture media and feed on growth of clone P/cSIN/92
The results in the previous sections show that HEK TF supported robust cell growth (Fig. 3), while the LV production was better when the cells were cultivated in HyCell TransFx-H (Fig. 4). Therefore HyCell TransFx-H was mixed with HEK TF or fed with HEK FS feed to broaden nutritional components and to exploit if there will be any synergetic or complementary effect between the media and feed from two different companies on the cell growth and also their potentials on the LV production in cultures induced at higher density. Data in Fig. 7 show that when the cells were cultured a mixture of HyCell TransFx-H and HEK TF (50% to 50%), the maximum viable cell density reached 8.85 × 106 cells/mL, higher than the maximum viable cell density, respectively, achieved in the HyCell TransFx-H (5.76 × 106 cells/mL) or HEK TF (8.11 × 106 cells/mL). Feeding the cells cultivated in HyCell TransFx-H with HEK FS feed increased the maximum viable cell density from 5.76 × 106 cells/mL to 9.97 × 106 cells/mL, resulting in a cell density increment of 4.21 × 106 cells/mL. The increment is much higher than the improvement of 0.68 × 106 cells/mL observed in the HEK TF culture fed with HEK FS (Fig. 3). Furthermore, when the cells cultivated in a mixture of 50% HyCell TransFx-H and 50% HEK TF were fed with HEK FS, the maximum viable cell density further increased to 11.1 × 106 cells/mL, the best cell density achieved in all conditions tested. The data in Figs. 7 clearly suggest a synergetic effect between the two media and also the feed in supporting the cell growth to high density.
High titer production of LV in clone P/cSIN/92 cultivated with HyCell TransFx-H, HEK TF and HEK FS feed at high cell density
Experimental design in this study took into consideration of previously obtained results (Figs. 3 and 7) and focused on effect of the following parameters on the LV productivity according to the scheme in Fig. 1: (i) growth medium, (ii) feed (HEK FS) used during the cell growth phase, (iii) medium exchange and (iv) cell density prior to induction. In these experiments, two cultures, respectively, grown in HEK TF and HyCell TransFx-H to a cell density of 1 × 106 cells/mL and induced without medium exchange were added. HyCell TransFx-H culture was as a reference while HEK TF culture was used for comparison. The data in Fig. 8a revealed that the LV volumetric productivity in the HyCell TransFx-H reference was 1.57 × 107 TU/mL while the titer from HEK TF culture was only 9.8 × 105 TU/mL. The LV productivity increased to a titer range between 6.4 × 107 and 11.1 × 107 TU/mL when the cultures were induced at a cell density of 5 × 106 cells/mL and with a medium exchange to fresh HyCell TransFx-H prior to the induction. This is a 4- to sevenfold increase compared to the titer in the HyCell TransFx-H reference culture. However, in the cultures induced at 5 × 106 cells/mL, but without a medium exchange to fresh HyCell TransFx-H, such improvement in LV productivity was not retained in the cultures grown in HEK TF or a mixture of 50% HEK TF and 50% HyCell TransFx-H. Only the culture grown in HyCell TransFx-H retained a high titer at 8.2 × 107 TU/mL, or fivefold improvement in the LV productivity even without a medium exchange. This result was very significant when compared to no more than twofolds of improvement obtained in the cultures using HyCell Transfix-H as growth media and fed with CB5 and other nutrients (Table 2). The fivefold improvement in the culture without a medium exchange before induction is also technically significant. This demonstrated that a procedure for medium exchange before induction is not necessary and implementation of this developed high cell density and high titer process is feasible at different scale bioreactors.
As illustrated in the experimental design in Fig. 1 for this set of experiment, the “quality” of the cells, respectively, grown in HyCell TransFx-H, HEK TF and a mixture of 50% HEK TF + 50% HyCell TransFx-H and fed with HEK FS was also assessed for supporting the LV production. The cells from various cultures (grown to 1 and 5 × 106 cells/mL in different media) were resuspended to fresh HyCell TransFx-H at a cell density of 1 × 106 cells/mL to have the same culture environment during the vector production phase. The data in Fig. 8b revealed the cell specific vector productivity was in a range of 14–22 TU/cell, and the difference was even smaller (between 15.5 and 17.3 TU/cell) among the cells from the cultures grown to 5 × 106 cells/mL. This result once again suggests the low vector production was mainly due to the nutrient limitation in the cultures grown in HEK TF or a mixture of 50% HEK TF + 50% HyCell TransFx-H and induced at high cell density. However, similar to that presented in Fig. 6, analysis of spent media from these cultures suggested the cell growth or virus production was unlikely due to the nutrient limitation of amino acids or glucose, or inhibited by lactate and ammonia.
Applicability of the developed feeding strategy to another stable clone for high titer production of LV at high cell density
The optimal feeding strategy developed in the previous section, namely growing the clone in HyCell TransFx-H, feeding the culture with HEK FS feed and then inducing the culture at 5 × 106 cells/mL without a medium exchange, was applied to clone P/SIN/18 for high titer production of LV. The clone P/SIN/18 was generated from HEK293 cell by a co-transfection, different from the transduction process used for P/cSIN/92. Data in Fig. 9 show the LV titer improved from 1.3 × 107 to 4.7 × 107 TU/mL, or a 3.5-fold improvement, when the cell density at induction was increased from 1 × 106 to 5 × 106 cells/mL in the cultures induced without medium exchange. However, it one again showed the high titer was only maintained in the culture grown in HyCell TransFx-H, suggesting the developed feeding strategy was able to provide nutrition requirements for two clones generated from HEK293 by different processes.
The results from this study demonstrated that the volumetric LV productivity could be improved by up to fivefold in shake flask cultures through nutrient optimization or balancing of culture media and development of feeding strategy. Although the conditions such as DO and pH in shake flask cultures were not controlled, oxygen supply (or DO concentration) was less likely to be a limiting factor for cultures induced at ≤ 5 × 106 cells/mL, as a cell density up to 20 × 106 cells/mL was achieved before in the shake flask culture conditions (agitation and culture volume) employed in this study. pH in the culture tended to decline with accumulation of lactate generated over the time course. Its effect on the LV production has not been tested yet. The high cell density process developed in this study was successfully scaled up to single bench scale (3L) bioreactor for high titer production of LV by other member in the team. Data will be published separately.