At Duoning, we provide DuoBioX^TM series cell culture bioreactors from process development to large scale manufacturing. The Explore series of benchtop bioreactors are positioned for upstream process development, optimization, and characterization at the laboratory stage, with volume ranging from 500 mL-15 L. The Pro series single-use bioreactors are production systems that meet the GMP requirements of commercial scale, with volume ranging from 50 to 2,000 L.

The Explore bioreactors are benchtop glass bioreactors with a distributed layout of satellite tanks, using centralized control, capable of supporting 0.6 - 15 L culture volumes. The software adopts advanced SCADA architecture, which can realize multi-parallel culture operation, data acquisition and result comparative analysis.

DuoBioX^TM Explore Parallel Bioreactors

Pro series single-use bioreactors adopt bottom stirring tank design, which can realize continuous scale-up from 50 L - 2,000 L. Combined with 3D single-use cell culture bags developed and manufactured by Duning, which feature good biocompatibility, and flexible manifold design, can meet all kinds of complex requirements in upstream processes. Pro bioreactors' mixing and sparging integrated design not only provides good mixing effect but also ensures excellent gas mass transfer performance.

DuoBioX^TM Pro Single-use Bioreactors

Scale-up consistency of cell culture process - For benchtop bioreactors in the process development stage, it is particularly important to provide reliable process parameters for subsequent scale-up, so 3 L – 5 L bioreactors are usually used as the starting point for the development of the cell culture process and as a tool for continuous optimization. In this study, the basic scaling principle of constant power consumption per unit volume (P/V) was used to investigate the consistency of the culture process from the Explore benchtop 3 L to the Pro 500 L, and the excellent scaling consistency between the two scales was proved.

CHO Cell Line

The cell line used in this study was recombinant CHO-K1 cell, and the product expressed by the cells was a bispecific antibody. CHO (Chinese Hamster Ovary) cells are the most representative mammalian expression hosts for genetically engineered proteins, and they are the most successful cells for expressing foreign proteins. CHO cells were able to grow to high density in serum-free suspension culture and maintain high levels of protein expression during extended cell culture.

CHO cells are the preferred manufacturing hosts for more than 70% of protein-based therapeutics, including monoclonal antibodies, recombinant proteins, and vaccines. At present, the commonly used CHO cell lines are: CHO-K1, CHO-DXB11, CHO-S, CHO-DG44, CHO-GS, etc.

Materials

Cell culture media

Basal media: Media C (also for seed train)

Feed media: DN feed 2, DN feed B2

Cell strain

CHO-K1

Bioreactor systems

DuoBioX^TM Explore multi-parallel bioreactor, 3 L

DuoBioX^TM Pro single-use bioreactor, 200 L & 500 L

Methods

Cell culture

For fed-batch mode, the initial culture volume is generally less than the maximum working volume of the tank, and the space left will be intermittently replenished several times during the culture process, so the culture volume will gradually increase until the end of the culture. After the cells are seeded, they consume nutrients (e.g., carbon sources, nitrogen sources) from the initial media for growth. With the continuous increase of cell density, the nutrients in the tank will be depleted, while the metabolic wastes continue to accumulate, but the nutrients required by the cell growth can be supplemented in time by feeding, so that the cell density will further increase. When the metabolic wastes accumulate to the point that the cell growth is significantly affected, the cell density and viability will also start to drop from the peak value.

pH control

pH down-regulation was performed by adding CO₂ into the culture through 1 mm sparge to lower pH.

pH up-regulation was performed by pumping base solutions such as Na₂CO₃, NaHCO₃ or NaOH into the culture through a peristaltic pump.

In general, the appropriate pH range for CHO cell culture is 7.0-7.2. In this study, the pH value of the culture was measured online through the pH sensor, and the flow rate of the CO₂ mass flowmeter and the speed of the base adding pump were automatically adjusted according to the measured value. The pH control dead zone was set to ±0.2, which could effectively preair supply pH adjustment overshoot.

DO control

The dissolved oxygen control of the culture system is realized by sparging different kinds of gases into the tank. Air and O₂ are sparged into the tank to increase DO, while N₂ can be used to control DO downregulation, achieving lower DO levels and even maintaining an oxygen-free environment. Due to the low solubility of oxygen in the media, its mass transfer efficiency is affected by the specific surface area of the bottom air supply and the contact time. The suitable range of dissolved oxygen for most mammalian cells is usually between 20% and 60%. The dissolved oxygen in the media is monitored online by an optical DO sensor, while the MFC-O2 flow rate is automatically adjusted according to the measured value.

In this study, the pore size of the sparger at the bottom of the Explore benchtop bioreactor was 1 mm, and the blade was a classic single-layer three-blade type with angle of 45°, which could generate downward axial flow and reduce the shear force on cells due to agitation. At the initial stage of the culture of Pro single-use bioreactor, 1mm macro-sparger bottom air supply was adopted, and the strategy was to combine macro-sparger and micro-sparger. When the VVM reached 0.3, turn on the 20 μm bottom micro-sparger as additional.

Feed control

Due to the fed batch operation mode adopted, regular feeding was required during the culture process. For the Explore benchtop bioreactor, the auxiliary simulation 4-20 mA input interface of the controller is used to connect the external scale, and for Pro bioreactors, they came with load cells, so for both bioreactor series, automatic feeding can be performed by built-in Feed In peristaltic pumps controlled by weight, and the feeding time point and frequency are programmed in the control software to run automatically.

Parameter setting and control strategy – fed-batch

Bioreactor configuration

Viable cell density and viability

Cell metabolism and protein concentration

Conclusion

According to the basic scaling principle of constant P/V, the fed batch culture process of CHO-K1 cells was successfully scaled up from a 3 L benchtop bioreactor to 200 L and 500 L single-use bioreactors, and the cell growth performance and protein productivity (>4 g/L) showed good consistency.

Information of related products

Cell culture media

Bioreactor