December 1, 2012 (Vol. 32, No. 21)
Process development and optimization is critical to enhancing the productivity of various cell lines. Nutrient requirements for peak productivity can be clone and process specific. Implementing the right feed formulation and strategy can greatly enhance the productivity of cell cultures in a fed-batch process.
A small-scale Design of Experiment (DoE) method was used to determine the optimal feed strategy (formulation, concentration, and timing) to maximize both product titer and peak viable cell density for several CHO clones. To alleviate concerns about media component variability, only chemically defined HyCell CHO medium and HyClone Cell Boost feeds were screened. Initial screenings were conducted at small-scale (1 mL cultures), allowing for a higher number of treatments and replicates. Once the optimal process for a particular clone was determined, scale-up to shaker flasks and benchtop bioreactors was performed, in order to compare batch and fed-batch product expression levels, and scalability of the process.
Materials & Methods
The experimental design included high-throughput methods at the plate level and/or shaker flask level with CHO clones derived from CHO-K1 origins. Various feed strategies were evaluated. All cultures were seeded with 2.5 x 105 cells/mL in HyCell CHO medium (Thermo Fisher Cat. # SH30934). Volumes were 1 mL at plate level, or 35 mL at shaker flask level. Cultures were supplemented with L-glutamine solution (Thermo Fisher Cat. # SH30034) to a concentration of 6 mmol/L. Cultures were incubated at 37ºC at 5% CO2.
Starting on day three or four, cultures received L-glutamine supplementation at 2 mmol/L and every other day thereafter. Selected feed conditions also began on day four and continued per their specific feed strategy until day 14 or until viability reached ≤ 50%. Viable cell density (VCD) and percent viability were determined on a daily basis using CellaVista or Vi-Cell XR instrumentation for plate and shaker samples, respectively. Protein production specific to the clones was analyzed starting day one or day five and every day thereafter using Octet instrumentation and applicable ELISA methods and analysis, depending on the protein analyzed.
The optimal product titer was determined for each cell clone and the correlating feed strategy that enhanced production (Figure 1). The cultures were then scaled up to bioreactors, both 10 L batch cultures and 7 L or 8 L fed-batch culture volumes. (Note: Lower volumes of 7 L and 8 L were used for fed-batch runs to accommodate feed volume additions during culture.) The bioreactors were seeded and supplemented identically to the screening studies. Fed-batch cultures were then treated with the particular clone’s preferred feed strategy. VCD, viability, and protein production were determined by the same methods (Figure 2). Nutrient and metabolite levels (glutamine, glucose, glutamate, lactate, ammonia, and pH) were monitored using a Nova Biomedical BioProfile FLEX instrument.
There are many Cell Boost products that complement HyCell CHO medium well in a fed-batch process. To ensure the best performance from each CHO clone, various feed formulations and strategies should be evaluated. We recommend evaluating feeds at numerous concentrations ranging from 2.5% v/v to as high as 10% v/v. Another factor to consider is the feed schedule for each clone. Using several high-throughput methods and small volume culture, we were able to evaluate daily and every other day feed schedules for each feed in the study with the various CHO clones. The more promising strategies were then scaled to benchtop bioreactors.
With the CHO Clone B evaluated, we observed an increase in titer using Cell Boost 2, 4, and 5. Feed concentrations of 50 g/L fed at 10% v/v were used. A schedule of every other day feeds was ideal for improving productivity in this cell clone. Of the top three performing feed strategies, the combination of Cell Boost 2 and 5 (60/40 ratio) fed every other day was selected for the scale-up fed-batch bioreactor run. This resulted in a 3.1x increase of product titer compared to the batch culture. Increases in both viable cell densities and productivity were observed.
Process parameter settings were not considered in this particular study, but are an additional step in creating the ideal fed-batch process to achieve the best recombinant protein production from each CHO clone.
CHO cells have become one of the most widely used cell lines in bioprocessing today. With such a variety of clones in use, it is important to optimize each cell culture strategy to get the best performance from each clone.
One of the goals of this study was to increase product titer of selected CHO clones by extending the culture time as seen above.
Using various feed formulations and strategies, both fed-batch cultures were extended by 2 and 3 days resulting in an increased product titer over batch culture. Future studies will include the modification of process parameters by adjusting volume, temperature, agitation, and dissolved oxygen settings in bioreactor cultures. These process parameter optimization steps, along with base medium selection and feed strategy design, will result in each CHO clone attaining its peak performance in culture.
Process optimization along with a well-founded, fed-batch procedure can result in higher product titer levels in a variety of CHO clones. This begins with a versatile basal medium such as Thermo Scientific HyCell CHO, which is chemically defined, and supports high cell densities and increased productivity for a variety of CHO clones. When paired with chemically defined process feed supplements such as Thermo Scientific HyClone Cell Boosts, a considerable increase in product titer is possible.
Thermo Fisher Scientific