Our ActiPro™ cell culture production medium is intended to be used in combination with Cell Boost™ 7a and 7b supplements to enhance recombinant protein production in fed-batch or perfusion processes. In this application note, we demonstrate the development of a fed-batch process for a Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb). Our results emphasize the importance of an appropriate feeding strategy to achieve optimal cell growth and productivity.
Introduction
We developed ActiPro™ cell culture medium and supplements to support high productivity of CHO cells in biomanufacturing of recombinant proteins, and the products have delivered excellent results with various CHO cell lines (1). ActiPro™ production medium and Cell Boost™ feed supplements are intended to be used together, following an easy-to-use starting protocol.
We designed the feed supplements to complement each other and the recommended ratio of Cell Boost™ feed supplements 7a to 7b is 10/1 (v/v). As a start, we recommend that feed volumes of Cell Boost™ 7a and 7b supplements are 3% and 0.3%, respectively, of the total culture volume. In previous experiments, this feeding strategy was also shown to be optimal for a high-titer mAb-producing CHO DG44 cell line (internal data). However, you should adjust the feed volumes with respect to the nutritional requirements of the specific cell clone used. In this application note, we developed a feeding strategy in shake flasks for a CHO-S cell line producing a monoclonal antibody in lower titers. We further optimized the productivity in bioreactor cultures using ReadyToProcess WAVE™ 25 bioreactor system.
Materials and methods
Cell lines
We performed studies with CHO-S cells (licensed from Cobra Biologics Ltd.). For shake flask cultures, we passaged the cells four times after thawing and cultured for 72 h prior to inoculation. For bioreactor cultures, we passaged the cells six times after thawing and cultured for 96 h prior to inoculation.
Batch cultures in shake flasks
We performed the batch cultures in 500 mL shake flasks filled with 100 mL ActiPro™ medium. Cells were inoculated at 0.3 × 106 viable cells /mL, and we maintained cultures in 37°C and 7.5% CO2 at a rocking speed of 105 rpm. We performed all experiments in duplicate and took samples daily - measuring for cell density, viability, productivity, and metabolite content.
Fed-batch cultures in shake flasks
We conducted fed-batch culturing in shake flasks were conducted to obtain an optimal feeding strategy. Studies were performed in 1000 mL shake flasks with a starting working volume of 250 mL ActiPro™ medium. We inoculated cells at 0.3 × 106 viable cells/mL, and starting on day 3, we added Cell Boost™ feed supplements once daily at volumes listed in Table 1. Glucose was added to the cultures when glucose concentration dropped below 2 g/L. We maintained the cultures in 37°C and 7.5% CO2 at a rocking speed of 105 rpm. We performed all experiments in duplicate, and samples were taken daily - measuring for cell density, viability, productivity, and metabolite content.
Table 1. Setup of shake flask experiment
| Shake flask | ActiPro™ medium starting working volume (mL) | Cell Boost™ supplements 7a/7b (% of working volume) | Cell Boost™ supplements 7a (mL) | Cell Boost™ supplement 7b (mL) |
| 1000 | 250 | 4.0/0.4 | 11 | 1.1 |
| 1000 | 250 | 3.0/0.3 | 7.6 | 0.76 |
| 1000 | 250 | 2.0/0.2 | 5.4 | 0.54 |
Fed-batch bioreactor cultures
We performed bioreactor cultures in the ReadyToProcess WAVE™ 25 system operated in dual mode. Two 10 L Cellbag™ bioreactor bags for the duplicate cultures were place on the same rocker. Culture parameters are listed in Table 2. We controlled cultures using the UNICORN™ system control software. We filled Cellbag™ bioreactor bags with 3 L ActiPro™ medium and inoculated to a cell density of approximately 0.3 × 106 viable cells/mL. Starting on day 3, we fed cultures with 2% Cell Boost™ supplement 7a/ 0.2% Cell Boost™ supplement 7b once daily, as was shown to be optimal in shake flask cultures. Starting on day 5, we maintained glucose concentration at 2 g/L in the cultures. The cultures were harvested on day 14. We took samples daily and measured for cell density, viability, productivity, and metabolite content.
Table 2. Culture parameters for 10 L fed-batch bioreactor cultures
| Starting volume | 3 L |
| Cellbag™ bioreactor | 10 L |
| Temperature | 37°C |
| Dissolved oxygen | 40% air saturation |
| pH | 7.1 |
| Rocking speed | 22 to 29 rpm |
| Supplementation
(from day 3) |
2.0% Cell Boost™ supplements 7a and 0.2% Cell Boost™ 7b |
| Glucose | Added on demand to maintain a concentration of > 2 g/L. |
| Harvest criteria | 9 days of feeding or when the cell viability dropped below 70%. |
Analytical methods
We measured cell density and viability, glucose, lactate, ammonium, glutamine, glutamate levels in cell suspension using a Bioprofile FLEX analyzer. The amount of produced mAb was measured in clarified culture feed with a CEDEX Bio analyzer. Glycoanalysis was performed by Cobra Biologics Ltd., Staffordshire, UK. Charge variant distribution was determined by cation exchange chromatography on a ProPac WCX-10 2 × 250 mm protein column.
Results
Shake flask cultures
Cell growth and productivity for shake flask batch cultures grown in ActiPro™ medium are displayed in Figure 1. To determine optimal feeding regimen, we supplemented shake flask cultures with Cell Boost™ supplements 7a and 7b in ratios of either 4%/0.4%, 3%/0.3%, or 2%/0.2% of total culture volume. Cell growth and productivity for the tested feed volumes are shown in Figure 2. Our results indicate optimal feeding at 2% Cell Boost™ supplement 7a/0.2% Cell Boost™ supplement 7b, giving the highest cell density and productivity for the selected cell line.
Fig 1. Viable cell density and productivity in shake flask batch cultures.
Fig 2. Viable cell densities and productivity in shake flask cultures supplemented with Cell Boost™ supplements 7a and 7b to varying volumes.
Fed-batch bioreactor cultures
Viable cell density (VCD) and productivity of the bioreactor cultures are shown in Figure 3. A peak VCD of 15 × 106 cells/mL and productivity of 1.6 g IgG/L were achieved, compared with the VCD of 12 × 106 cells/mL and productivity of approximately 1.2 g IgG/L achieved in shake flasks. Metabolite concentrations are displayed in Figure 4, showing the concentration profiles for glutamine, glutamate, ammonium, glucose, and lactate during the culture. Figures 5 and 6 depict the product quality attributes (charge variant distribution and glycoprofiles) for day 12, 13, and 14. Generally, we observed only subtle changes in the quality attributes during the last three days of the culture. Shake flask culturing is a good tool for screening of certain culture conditions. However, in many cases, bioreactor systems offer improved growth conditions over culturing in shake flasks, as the culture parameters can be more accurately controlled. Therefore, as expected, both cell growth and productivity were further improved in the bioreactor fed-batch cultures.
Fig 3. Cell growth and productivity in the fed-batch bioreactor culture.
Fig 4. Metabolite concentrations in the fed-batch bioreactor culture.
Fig 5. Charge variant distribution in the fed-batch bioreactor culture.
Fig 6. Glycoprofile of the fed-batch bioreactor culture.
Conclusions
In this application note, we show the development of a fed-batch process for a low-producing CHO cell line, using ActiPro™ culture medium and supplements. We assessed feed ratios of 2.0%/0.2%, 3.0%/0.3%, and 4.0%/0.4% Cell Boost™ supplements 7a to 7b. For this cell line, we found that optimal cell growth and productivity was obtained at 2% Cell Boost™ supplement 7a and 0.2% Cell Boost™ supplement 7b. Our results reflect the importance of determining appropriate feeding regimen based on the nutritional requirements of a specific cell line, to avoid over or under feeding of the culture. As charge variants and glycoform distribution can vary significantly between and during the cultures depending on selected feeding strategy, you should consider these important product quality parameters when selecting feeding strategy and scaling up the process.
Reference
- (1)Application note: Scale-up of CHO cell fed-batch cultures in HyClone ActiPro medium supplemented with Cell Boost 7a and 7b.
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