Everything you ever wanted to know about cell culture process transfer

The typical process cycle

A specific product is usually produced by thawing the cells, cultivating these up to a certain amount of cell density in incubated shakers, followed by then transferring the process into a bioreactor. To increase the amount of product, the bioprocess is accordingly finally scaled-up to different unit sizes. However, such a sequence of steps is easier said than done. Many different factors, biological as well as physical parameters, need to be considered for a successful process transfer. Therefore, this post is aimed at shedding light on these.

Be aware of critical factors for process transfer

A process transfer combined with a scale-up step from shake flask to bioreactor is not always easy and smooth. The environment for the cells changes totally, starting from the aeration to the cultivation method itself. The environment for the cells changes totally, starting from the aeration to the mixing method itself. The fact that the cells are not being shaken anymore and rather being stirred results in a change of mixing within the bioreactor. This dramatic change for the sensitive mammalian cells leads to shear stress and foaming issues, but nowadays these are quite well controlled due to the addition of antifoam and cell surfactants (Xing, Kenty, Li, & Lee, 2009). Besides the observations mentioned above, few more critical engineering parameters exist that need to be considered:

1. Oxygen transfer:

Since mammalian cell culture processes are aerobic, the oxygen transfer into the cells is a very critical factor. To ensure adequate oxygen availability, the agitation speed, gas flow rate and gas composition need to be controlled. As mammalian cells do not have a cell wall, the power inputs may need to be kept low, leading to inhomogeneities within the culture (Croughan, Hamel, & Wang, 1987).

2. Culture media mixing:

Another important influential parameter is the mixing within the cell culture broth which needs to be considered.  Hence, insufficient mixing results in pH and nutrient gradients, which has been shown to dramatically influence cell growth and antibody production (Bylund, Collet, Enfors, & Larsson, 1998; Osman, Birch, & Varley, 2001).

3. CO₂ accumulation:

Due to the low power input and aeration rates, the CO₂ concentration in the culture media is elevated, which negatively impacts product quality as well as cell growth and protein production (Gray, Chen, Howarth, Inlow, & Maiorella, 1996).

The scaling-up recipe

Even today, scale-up is an erratic procedure since bioprocesses are not properly monitored and understood in detail. Two very important process relevant parameters are agitation and aeration. On the one hand, agitation is set to achieve homogenous mixing and oxygen transfer into the cells. Aeration, on the other hand, is regulated to supply the cells with sufficient amount of oxygen as well as to eliminate excess amount of CO_2. Especially in large-scale bioreactors, excess CO₂ concentration can pose a serious problem. Therefore, it is important to consider the aeration and agitation rates, position of the impeller and sparger, which will affect bubble size (Gray et al., 1996). The specific engineering parameters which are needed to successfully execute the scale-up process are discussed below at length (derived from DECHEMA, Society of Chemical Engineering and Biotechnology).

1. k_La – The volumetric mass transfer coefficient

The k_La coefficient denotes the efficiency of gas transfer from the gas phase into the liquid phase. It is dependent upon the design and operating conditions of the bioreactor. Moreover, it is also influenced by the impeller design, aeration and agitation rates. The higher the k_La value, the greater the aeration performance of the cultivation system. Several methods exist to determine this coefficient with a corresponding dissolved oxygen probe. Some of the most prominent approaches are as follows.

• Dynamic gassing out technique
  In the initial step, the bioreactor system is in a stationary state, which is disrupted by a sudden stop of oxygen supply. This causes a decrease in dissolved oxygen concentration through its consumption by the cells. After the oxygen is consumed, the supply is switched on again and the gas returns to its original .
• Static gassing out technique
  In this technique, the oxygen is gassed out of the liquid after addition of nitrogen. Afterwards, the liquid is stirred and aerated again, and the increase of dissolved oxygen is monitored.
• Sulphite technique
  This method is based on the reaction of sodium sulphite with oxygen, where the sulphite is oxidized with the help of metal ions to sodium sulphate. The consumption of oxygen then determined.
  

For the determination of the k_La coefficient (h-1), besides the oxygen transfer rate, OTR (mg O₂*L-1*h-1), the equilibrium oxygen concentration, cO₂* (mg/L) and the dissolved oxygen concentration cO₂ (mg/L) need to be known.