What to Consider When Scaling-Up Your Bioreactor?

Co-authored by Shahin Heshmatifar and Marie-Laure Collignon.

Single-use stirred tank bioreactors have become a popular choice for large-scale cell culture of suspension cell lines and commercial manufacturing of therapeutics produced by these lines. With numerous bioreactors available in the market, covering a wide range of working volumes,from milliliters to thousands of liters;processes are developed using different technologies at various scales. There are two core engineering challenges to be addressed: technology transfer (tech transfer) and scale-up. 

Scale-up and tech transfer activities pose several challenges, including defining operating conditions, such as agitation rate and gas sparge rate. However, these operating parameters are non-scalable. Selection of non-scalable parameters will result in different physical and chemical environmentsthat the cells reside in. For example, oxygen delivery and mixing will be different at different scales. It is imperative to determine the critical environmental parameters that impact the biological attributes, such as product yield, cell growth and quality. Additional critical environmental parameters include oxygen transfer capacity, shear stress and mixing. Whilst these parameters cannot be measured directly, there are engineering tools available to measure and monitor these parameters indirectly. 

These engineering tools can be employed to define the bioreactor conditions and scale-up the process.The most commonly used tools arepower input per unit volume (P/V), oxygen transfer rate (kLa) and mixing time.

P/V can be identified as the rate of impeller energy transfer into the cell culture media. It relates to many engineering parameters such as homogenization and gas-liquid transfer. It is also a good indicator of shear stress experienced by the cells. A recent approach to evaluate potential risk of negative impact on the biological attributes is based on the comparison of the cell size with the main turbulent flow structures.This risk can be predicted by evaluating the smallest size of the turbulent flow structure with the P/V magnitude.

Determination of the volumetric oxygen mass transfer coefficient (kLa O2) is regarded as a standard benchmarking method for assessing the ability of the bioreactor system to transfer oxygen from the gas to liquid phase. Oxygen mass transfer (OTR) is critical for aerobic cultures and can be challenging, as oxygen is the least soluble and most quickly consumed nutrient by cells, therefore this typically becomes the rate limiting factor in high cell density cultures. Oxygen transfer rate should be equal or above oxygen uptake rate (OUR).kLa can be measured experimentally or can be predicted by using the Van’Reit correlations involving the P/V and the superficial gas velocity.

Mixing time is defined as the time required to reach 95% fluid homogenization following deliberate disturbance to the stability, such as by addition of base. Short mixing times are essential for a successful cell culture. Long mixing times are undesirable, as in the case of frequent chemical additions, such as base for pH control; they imply longer period of contact between cells and possibly harmful chemicals. Although good mixing can be achieved relatively easily at smaller scale cultures, when scaling to larger volumes, rapid mixing can become more of a challenge. The mixing time can also be measured experimentally or predicted using a correlation involving P/V and geometrical parameters.

It is not possible to maintain all the scalable parameters when scaling-up or performing a technology transfer.Physical rules linking scaling parameters to non-scalable parameters follow different formulae; the mathematical relations between the parameters results in change once a constant is kept. It is easier to scale-up in bioreactors of different sizes that are proportional and geometrically similar. Selecting one of the scalable parameter depends on its impact on the engineering parameters (mixing time, shear stress, oxygen transfer rate) and finally biological attributes, such as cell growth, product yield, and quality.

The success of scale-up depends on sound engineering and scientific characterization of the bioreactor system, including engineering parameters such as kLa, P/V, mixing times and shear stress distribution.

Learn more about Controlling the Key Parameters of a Bioreactor.

Co-authored by Shahin Heshmatifar and Marie-Laure Collignon.