In this application note, we compare cell growth and recombinant protein production of multiple Chinese hamster ovary (CHO) cell clones when cultured in HyClone™ ActiPro™ basal medium and HyClone™ Cell Boost™ 7a and HyClone™ Cell Boost™ 7b feeding supplements.
The results from shake flask cultures showed high viable cell densities and protein production. In addition, we demonstrated the scalability of the culture process in 2 and 50 L bioreactor cultures. We also found that cell density and protein production were comparable between scales. Protein yields reached up to 5 g/L in shake flasks, and up to 5.5 g/L in bioreactor cultures.
Introduction
The most widely used cell lines for bioprocess production of therapeutic proteins originate from Chinese hamster ovary cells. These cells are robust in culture and can produce a variety of recombinant glycoproteins at high levels in large scales. We designed and optimized our ActiPro™ medium and Cell Boost™ 7a and 7b supplements for large-scale protein production in fed-batch suspension culture, using recombinant CHO cells.
Chemically defined with no peptides or hydrolysates, ActiPro™ medium and Cell Boost™ supplements are animal-derived component-free (ADCF™) and manufactured using current good manufacturing practices (cGMP). As ActiPro™ medium and Cell Boost™ 7a and 7b supplements do not contain hypoxanthine or thymidine (HT), their formulations support the dihydrofolate reductase (DHFR) gene amplification and selection system.
ActiPro™ medium and Cell Boost™ 7a and 7b supplements can be used together in a fed-batch process, or the medium and supplements can be used in combination with other products to increase productivity of an existing process. We demonstrate the growth and productivity of CHO cells cultured in ActiPro™ medium and Cell Boost™ 7a and 7b supplements.
We performed studies with multiple CHO cell clones in 30 mL shake flask cultures. To demonstrate scalability of the process, we transferred cultures to 2 L and 50 L bioreactor cultures and cell growth and productivity of selected clones across both scales.
Materials and methods
Studies in shake flask cultures
We recovered three different proprietary CHO cell clones CHO-S (monoclonal antibody [mAb] producer), DG44 (mAb producer), and CHO-S (parental line, nonproducer) from cryopreservation according to standard protocol and subcultured every third or fourth day. Once we had recovered the cells, we inoculated them in ActiPro™ medium. A minimum of three adaptation passages were completed until the cells adapted. Cells were considered adapted when their doubling time was less than 24 h.
Following adaptation, we seeded the cells into 30 mL volume in shaker flask at a density of 0.5 × 106 viable cells/mL. Starting on day three, we fed each culture Cell Boost™ 7a feeding supplement at 3% of vessel volume and Cell Boost™ 7b supplement at 0.3% of vessel volume. We maintained the culture at a minimum of 3 g/L of glucose by supplementing with a 250 g/L glucose concentrate as determined, using Accutrend Plus glucose monitor. We performed all studies in duplicate.
2 L bioreactor cultures
We expanded CHO-S (mAb producer) and DG44 (mAb producer) cell clones in 2 L bioreactor cultures.
We filled the bioreactor with 2 L of ActiPro™ medium and equilibrated to 37°C and pH 7.0 before seeding with 0.5 × 106 cells/mL.
Starting on day three, we fed each culture Cell Boost™ supplement 7a at 3% of vessel volume and Cell Boost™ feeding supplement 7b at 0.3% of vessel volume. Glucose was maintained at 3 g/L as measured with BioProfile FLEX analyzer.
Cultures were run in chemostat mode: each day prior to feeding, the culture volume was drained to 2 L total volume. Antifoam was added as needed to minimize foaming. Table 1 lists the operating parameters and culture conditions.
Table 1. Operating parameters and conditions for 2 L bioreactor cultures
|
Medium |
ActiPro™ growth medium with 6 mM L-glutamine |
|
Supplements |
Cell Boost™ supplement 7a (3% of working volume) Cell Boost™ supplement 7b (0.3% of working volume) |
|
Culture chamber |
2.5 L bioreactor |
|
Mixing |
350 rpm |
|
Gas flow |
Set by controller to maintain 50% dissolved oxygen (DO) |
|
Seed cell concentration |
0.5 × 106 viable cells/mL |
|
pH set point |
7.0 |
|
DO set point |
50% |
|
Antifoam |
Antifoam C emulsion as needed |
|
Working volume |
2 L chemostat operation |
|
Harvest criteria |
Culture viability < 80% |
50 L bioreactor cultures
Due to its high level of protein production, we chose the DG44 (mAb producer) cell clone for expansion in 50 L Xcellerex™ XDR-50 bioreactor cultures.
We seeded the cells at 0.5 × 106 cells/mL into a starting volume of 25 L of ActiPro™ medium. Starting on day three, we fed the culture Cell Boost™ feeding supplement 7a at 3% of the current volume, Cell Boost™ feeding supplement 7b at 0.3% of the current volume, as well as glucose to maintain a level at 5 g/L.
Antifoam C was added as needed to prevent foaming. We fed the cultures to a final working volume of 50 L.
Table 2 lists the operating parameters and culture conditions.
Table 2. Operating parameters and conditions for 50 L bioreactor cultures
|
Medium |
ActiPro™ growth medium with 6 mM L-glutamine |
|
Supplements |
Cell Boost™ supplement 7a (3% of working volume) Cell Boost™ supplement 7b (0.3% of working volume) |
|
Culture chamber |
50 L bioreactor |
|
Mixing |
95 rpm |
|
Gas flow |
Set by controller to maintain 50% dissolved oxygen (DO) |
|
Seed cell concentration |
0.5 × 106 viable cells/mL |
|
pH set point |
7.0 |
|
DO set point |
50% |
|
Antifoam |
Antifoam C emulsion as needed |
|
Working volume |
Fed-batch operation Start: 25 L Final: 50 L |
|
Harvest criteria |
Culture viability < 80% |
Results and discussion
Studies in shake flask cultures
Before we set up all cell clones for terminal growth curves in fed-batch mode, we adapted the cells in culture medium (see Fig 1). Viable cell densities were measured daily, and samples for productivity determination were taken.
Two of the clones we used produce mAbs, and one is a parental line that does not produce recombinant protein. Results are shown in Figures 2 to 4.
Fig 1. Comparison of DG44 (mAb producer) cells (adapted after eight passages), CHO-S (mAb producer) cells (adapted after four passages), and CHO-S (nonproducer) cells (adapted after three passages) grown in ActiPro™ medium.
Fig 2. Growth and productivity of CHO-S (mAb producer) cells cultured in fed-batch mode. Viable cell density reached more than 30 × 106 cells/mL and IgG production reached 2.2 g/L.
Fig 3. Growth and productivity of DG44 (mAb producer) cells cultured in fed-batch mode. Viable cell density reached 20 × 106 cells/mL by day 8 and IgG production reached 4.3 g/L.
Fig 4. Growth of CHO-S cells cultured in fed-batch mode. Viable cell density reached more than 24 × 106 cells/mL by day 7. No productivity data is shown, as the CHO-S clone used here is a parental clone and does not produce recombinant protein.
Bioreactor cultures
Scalability of selected CHO cell clones was demonstrated in both 2 L and 50 L bioreactor cultures for the DG44 (mAb producer) cell clone.
For the 2 L bioreactor cultures, both cell clones reached a viable cell density of greater than 20 × 106 cells/mL by day 6 (Fig 5A). The DG44 (mAb producer) cell clone is a higher IgG producer than the CHO-S (mAb producer) cell clone; however, both reached production levels as high as expected for these clones and protein production continued for the length of the runs (Fig 5B).
Fig 5. (A) Viable cell density; (B) productivity of DG44 (mAb producer) and CHO-S (mAb producer) cells in fed-batch bioreactor cultures.
For the 50 L bioreactor culture, cell density reached nearly 25 × 106 cells/mL by day 7 and productivity reached 5 g IgG /L by day 10 (Fig 6). Metabolite levels for the 50 L bioreactor culture are shown in Figure 7. As shown, the daily increase in glucose consumption correlates well with the cell density in the log phase of growth. We also supplemented glucose to maintain levels required for cell growth.
Fig 6. DG44 (mAb producer) cell growth and productivity in 50 L ActiPro™ bioreactor cultures.
Fig 7. Metabolite concentrations for the 50 L bioreactor culture.
Conclusion
- We illustrate growth and productivity of multiple CHO cell clones cultured in ActiPro™ medium supplemented with Cell Boost™ feeding supplements 7a and 7b with regard to their viable cell density and protein production in shake flask cultures.
- When transferring the cultures from shake flask to 2 and 50 L bioreactor cultures, we found that selected clones showed similar performance to the shaker flask cultures.
- Both ActiPro™ medium and Cell Boost™ feeding supplements 7a and 7b supplements are versatile cell culture platforms that can be used with multiple CHO cell clones in both small and larger-scale applications.
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