Bioreactor systems rely upon the introduction of oxygen and carbon dioxide to both feed cell cultures and maintain a balanced pH for an optimised environment and controlled toxicity.
Numerous bioreactor components and features are critical for process optimization, including impellers for gentle mixing, baffling, spargers for gas introduction and the bioreactor shape. Here we focus on gas flow control in tandem with the use of spargers within bioreactors and discuss both technological challenges they face and possible solutions.
The introduction of CO2 needs to be extremely precise, repeatable and scalable. Traditional gas introduction has utilized thermal mass flow controllers however these are calibrated on air and supplied with a conversion factor for CO2. This introduces significant measurement uncertainty with a single conversion factor having an error approaching 2.7%. Furthermore, this conversion factor is neither linear across the measured scale of the device nor consistent across varying pressure and temperature regimes. It is therefore not surprising that many scaling issues are directly linked to this lack of precision within CO2 measurement and control.
Alicat mass flow controllers use a laminar flow element (LFE) within the heart of their sensor. This means that no conversion factors are required and the 0.6% of Reading accuracy remains valid regardless of the gas type being measured. Furthermore, this technology ensures a 10,000:1 measurement range such that a 100 SLPM device can measure down to 0.01 SLPM resulting in far fewer MFC’s per installation.
Smaller bioreactors are extremely effective at distributing oxygen and stripping carbon dioxide without resorting to spargers provided that environmental/physical conditions remain constant. However, this becomes inadequate for larger bioreactors, where the lower surface-area-to-volume ratios lead to carbon dioxide accumulation and prevent oxygen penetration. Therefore if we are going to remove carbon dioxide and introduce oxygen, Spargers are considered to be vital to the process.
Systems with both micro- and macro- spargers are useful because they are designed to cope with a multitude of applications with widely varying process conditions. It is known that macro-spargers create larger gas bubbles that are more effective at removing dissolved CO2 from solution. The compromise however is that the larger bubbles require much more robust agitation to break them up and release oxygen. This agitation might be acceptable for stronger cell lines but more fragile cell lines such as mammalian cells will themselves be broken up as well. In practice a macro-sparger with much lower power can be used to remove CO2 and then a parallel micro-sparger can introduce finer bubbles that are more efficient at delivering the required concentration of oxygen.
Bubble formation and size have a significant impact upon the dispersion of oxygen throughout the bioreactor. The nature of the sparger and how it is used have a direct impact upon this bubble formation such that gas pressure, the flow rate, the relative properties of the gas and liquid, sparger material, pore size and the distribution of the pores are all important. Micro-spargers tend to create smaller, spherical bubbles whilst macro-spargers tend to create bubbles that are a bit larger and are more unevenly shaped.
The amount of shear stress that the cell line will be exposed to is, in part, determined by the shape and size of the bubbles. Furthermore, the effectiveness of oxygen mass transfer and CO2 stripping is also determined by bubble formation. The optimisation of spargers is therefore important to ensure oxygen bubbles are evenly distributed and are evenly sized to enhance gas transfer whilst minimising cell destruction. In reality the variability seen in flow rates and the huge variation in vessel size makes the prediction of bubble characterization very difficult indeed. Such unpredictable behaviour makes it almost impossible to pre-determine the shear forces involved and thus the final oxygen mass transfer.
Pulse modulated sparging provides a method to control both bubble size and speed of release into the liquid media. An MFC with extremely low controlled flow rate (such as the Alicat BIO) ensure a slow build-up of pressure within the porous sparger disc. At such time that the pores are all filled up, there is a gentle release of bubbles into the bioreactor. It is essential that a high degree of resolution is available for flow rate adjustment as this determines the exact rate of release whilst maintaining uniform bubble size. Alicat mass flow controllers provide the lowest flow rate available in the market with the extremely stable zero and noise-free signal ensuring system stability and repeatability. Laminar Flow Element technology with its accuracy and precision ensures scalability across vessel sizes with a predictable oxygen transfer rate that is proportional to the predictable, exact gas flow rate. The linear scalability enables the user to confidently increase oxygen or air mass flow rates to support larger scale cell cultures.
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