The use of scale-down models is a common practice in the pharmaceutical industry to ensure the performance of particular production systems. The adoption of small-scale systems typically reduces costs and time expenditures in contrast to full-sized prototypes. This concept is widely established in process development, process characterization, process troubleshooting, and raw materials testing experiments.
A standard scale-down model for the manufacturing of recombinant proteins using CHO cells is a 2 L glass bioreactor. Although the key process parameters are comparable between scales, the use of 2 L bioreactors still remains resource and time consuming. Therefore, an alternative system that provides process comparability and ease of use is essential.
As such, scientists at Roche recently evaluated the performance of Dasgip’s 1 L cell-cultivation system (Figure 1) for use as a potential scale-down model.
The Dasgip system uses eight spinner vessels. The spinners are equipped with sensors for pH and DO, a marine impeller, a sparger, and a 0.22 µm vent filter for exhaust gases. The pH level is controlled via NaHCO3 and CO2 gassing.
DO is maintained at a constant level via oxygen enrichment of air and mass-flow adjustment of the total aeration by the MX4/4 controller. The Dasgip MX4/4 is composed of one mass flow controller (MFC) controlling the air, O2, and CO2 flows for each vessel.
A fixed total flow (air + O2) strategy is used to fulfill the cells’ oxygenation need and set to a maximum value to maintain the DO to the defined set point. Oxygen limits in the culture are avoided by automated addition of O2, with the flow only limited to the size of the MFC.
The homogeneity of each suspension culture is maintained via magnetic stirrers below the vessels. These stirrers are individually controlled by the SC8 module. To control the temperature, the vessels and stirrers are located in an incubator.
A successful scale-down/scale-up approach requires the transfer of level and control strategies for scale-independent parameters such as pH, DO, and temperature from manufacturing- to model-scale. Then a technical characteristic number is chosen to transfer the remaining parameters; in this study, the volumetric power input was selected.
In accordance with the 2 L scale-down model, the transfer of a constant volumetric power input was applied to the Dasgip system as well. The Table shows the corresponding parameters for the Dasgip system and the manufacturing scales.
To establish a reliable scale-down model, particular attention must be paid to the choice of the right aeration system. Accordingly, three different spargers (open steel pipe, L-sparger, and sintered microsparger—Figure 2) were evaluated regarding their influence on the cell culture’s performance in comparison to the manufacturing scale.
Dasgip spinners were inoculated with cells cultivated under selective pressure. After medium exchange by centrifugation, they were resuspended in 250 mL of production cell culture medium. The production step was performed as a fed batch culture and lasted approximately 14 days. To improve antibody production the process included a temperature downshift to slow down the cell metabolism while supplying the cells with enough nutrients through batch feed or glucose additions.
To monitor cell growth and metabolite levels, cell culture samples were taken and measured off-line. Back-up samples are centrifuged and analyzed using an IgG-HPLC assay with a protein A affinity column for antibody titer determination.
At the end of the production stage, the cells were harvested and separated from the cell culture medium. The harvested cell culture fluid was then further purified through a protein A affinity chromatography step to assess product quality.
The antibody charge distribution (acidic, basic variants, and main peak) was measured using ion-exchange chromatography. In addition, the formation of aggregates was evaluated using size-exclusion chromatography.