Cell Culture Scale-Up Using Stirred-Tank Single-Use Bioreactors

In bioprocess development, the working volume in the R&D phase is typically small and increases as development progresses. During cell culture scale-up, process performance optimized at small scale needs to be reproduced at larger scale, ideally without much need for process optimization at large working volumes. In this article we discuss, how we characterized the BioBLU^® c Single-Use Bioreactor portfolio to streamline cell culture scale-up and demonstrate, how we scaled-up the working volume of a cell culture process for mAB production from 3 L to 150 L, using single-use bioreactors at all stages.

When thinking about how to scale-up a cell culture bioprocess , scientists often decide to keep one parameter constant across scales, like the tip speed, k_La, or power input per volume.

Whichever scale-up strategy the investigator chooses, two important considerations need to be taken into account. First, the tip speed range, k_La range, and the P/V range, respectively, of the differently sized bioreactors need to overlap to some extent to be kept constant across scales. Second, information on certain bioreactor engineering parameters is required to define this overlap. This is why we have investigated the scale-up capabilities of Eppendorf BioBLU^® Single-Use Bioreactors for cell culture applications for culture scale-up from small to pilot scale.

Bioreactor geometries

BioBLU^® Single-Use Bioreactors from small scale to pilot scale are of geometrically and proportionally similar stirred-tank designs (Table 1).

Identifying a scalable tip speed zone

Tip speed is an important parameter for cell culture scale-up. Scale-up based on maintaining constant tip speed is one of the common strategies, especially for cultures sensitive to shear force damage. We identified 0.3 to 0.7 m/s as the tip speed scalable zone of the BioBLU c Single-Use Bioreactors, i.e. the tip speed range that all BioBLU^® c Single-Use Bioreactors can reach (Table 2).

Identifying a k_La-based scalable zone

Oxygen transfer is one of the most critical scale-up parameters for bioreactors, and it can be represented in the form of the volumetric mass transfer coefficient, k_La. When oxygen transfer is the rate limiting factor for a cell culture process, maintaining constant k_La can achieve scalable yield from small to large scale. For proper cell culture scale-up, it is important to select equipment of different sizes with similar k_La capabilities that offer sufficient overlapping so that the small scale success can be replicated in large scale.
As an example, we determined the k_La of differently-sized BioBLU^® c Single-Use Bioreactors (BioBLU 0.3c, 3c, and 50c) using the static gassing-out method [1] and identified a k_La-based scalable zone between a k_La of 1 and 6 h^-1 (Figure 2). k_La values in this range can be reached by all BioBLU^® c Single-Use Bioreactors tested and therefore replicated across scales.

Find more information on dissolved oxygen (DO) control in bioreactors .

Identifying a P/V-based scalable zone

Maintaining a constant impeller power consumption per liquid volume (P/V) between bioreactors is one of the most prevalent strategies for scale-up. To calculate it, the impeller power number Np (also known as Newton number, Ne) is needed. Np is a dimensionless number associated with each type of impeller. However, the measurable power numbers vary depending on various factors of the bioreactor set-up. The industry standard method to determining Np is to measure the impeller torque by using a rotational torque sensor.
We have determined the power number for the differently sized BioBLU^® Single-Use Bioreactors according to a protocol recommended by the DECHEMA^® [1] (Table 1). Using the Np values, it is possible to identify a scale-up zone, within which P/V values can be achieved by the bioreactor types of interest by applying a tip speed which is within the technical specification of the bioreactors. Figure 3 shows exemplarily the correlation of P/V and tip speed for the BioBLU^® 0.3 c, 3c, and 50 c Single-Use Bioreactors, respectively.

In a proof of concept study we developed a CHO cell culture scale-up workflow, which uses single-use bioreactors at all stages from bench to pilot production scale. For the scale-up strategy we selected the option of maintaining a constant P/V. To simplify cell culture scale up we used the Scale Up Assist software feature of the BioFlo^® 320 and BioFlo^® 720 bioreactor control stations from Eppendorf.

Scale Up Assist software feature for simplified cell culture scale-up
Both P/V and the impeller tip speed depend on the agitation speed. To keep P/V or tip speed constant when transferring the bioprocess to a differently-sized bioreactor, the agitation speed needs to be adapted accordingly. The Scale Up Assist software (Figure 4) feature from Eppendorf simplifies this process. The software is auto-populated with the power numbers and impeller diameters of various bioreactor types. The scientists only need to select the desired bioreactor types from a list and to enter the working volumes. The software then calculates the agitation speed which results in a defined P/V value or tip speed at the selected working volume. The Scale Up Assist is a software feature of the BioFlo^® 320 and BioFlo^® 720 bioreactor control stations from Eppendorf.

Comparable cell growth and antibody production from 3 L and 150 L
We scaled-up a CHO cell culture bioprocess for mAB production from 3 L to 10 L in BioBLU^® Single-Use Bioreactors and further up to 40 L and 150 L in Thermo Scientific HyPerforma SUBs, controlled by a BioFlo^® 320 and BioFlo^® 720 controller, respectively. By using the parameters calculated by Scale Up Assist feature and described in detail in our application note, we could match the growth profiles across all platforms in multiple batch runs with working volumes between 3 L and 150 L. All runs yielded similar IgG production values when they reached completion.

References:

[1] DECHEMA Expert Group Single-Use Technology; Recommendations for process engineering characterization of single-use bioreactors and mixing systems by using experimental methods (2nd edition); 2020 https://dechema.de/en/Single_Use_PE_Characterization-path-123211,124930.html

Cell Culture Bioprocess Scale-Up Workflow from Bench to Pilot/Production Scale using Single-Use Bioreactors