This is a quick introduction to sterilisation. We will go through this sterilisation in more detail in its own post, but since it is quite complicated this will just be a brief explanation so you understand the concept.
What is sterilisation?
Sterilisation is the removal or prevention of contamination at any point throughout the process.
Why do we need sterilisation?
Contamination is a big no-no in the industry. If your cell culture is contaminated, you must discard it. This is because contaminants can cause danger to the end user of the product. If your product is contaminated with bacteria and someone ingests/ injects it, they could get seriously sick. Therefore regulatory bodies require that there is no contamination present at any point of the process. Also, contaminants can compete with your cell line for nutrients and oxygen in the broth, which will reduce cell numbers, and could stop production of your product.
Levels of sterilisation required
No sterilisation: Process does not need to be sterile. This is mainly for processes that do not create a product that will be ingested i.e. Waste water treatment.
Protected fermentation: For processes that require some degree of sterility but do not tend to get contaminated if treated properly i.e. disinfection of needles, pasteurisation of milk.
Unprotected fermentation: Require a high degree of sterility due to high likelihood of contamination if exposed to the external environment. This goes for bioprocesses that grow cells in media since media contains many components (nutrients, oxygen, etc.) that are highly favourable by contaminants i.e. bacteria.
Examples of contaminants
Bacteria
Viruses
Mycoplasm
Fungi
How do you avoid contamination?
Bioreactors usually operate as closed systems, meaning they do not exchange any material with the environment around it. Here are some methods of preventing contamination in reactors:
Sterilise the reactor and any ancillary equipment (parts that you attach to the reactor)
Sterilise whatever is going into the reactor (air, buffers, media, nutrients, etc.)
Introduce a pure inoculate (cell culture) and feed. Before introducing the cells into the reactor make sure they are not contaminated (using a viable cell count to detect unusual growth). The feed should also be sterilised before introducing it to the reactor.
Maintain an aseptic operation. Make sure the bioreactor is properly sealed and there are no cracks or holes that expose it to the external environment.
Methods of sterilisation
Inactivation/ killing of viable microbes
This can be done by:
Heat treatment (heating the components to a high temperature to kill contaminants)
Irradiation
Chemical treatment (using strong chemicals i.e. disinfectants to kill contaminants)
Physical Removal of microbes
This can be done using filtration or centrifugation.
If you have any questions, please leave a comment below and I will get back to you ASAP!
In order to successfully expand cells without any trouble, a reactor must meet these requirements:
Operate aseptically: The reactor must be sealed and sterilised so that contamination will not occur during the process. This is important as contaminants not only compete with your cells for nutrients but also can harm your customer if it is in the final product. Contaminated cell cultures must be discarded according to regulatory bodies.
Agitate and aerate effectively: Cells need oxygen to survive, hence effective aeration is needed. Effectively agitated broths produce homogeneous flow: where all the liquid has the same concentrations of oxygen, nutrients, pH buffer, etc. This is important as we want all cells to be exposed to the same conditions so that they grow and proliferate in the same way e.g. if a region of the liquid has too little oxygen all the cells in that region will die.
Control: We must be able to control the conditions in the reactor (temperature, pH, air flow rate) not only so that the cells are growing in conditions we know optimise survival and growth, but to ensure that we have an idea of what conditions the cells are in and therefore predict how are cells will proliferate and grow.
Minimise evaporation: We don’t want the liquid in the reactor evaporating since it contains cells and useful nutrients. Evaporation also means that our predictions of what is happening inside the reactor won’t be accurate.
Allow monitoring/ sampling: So that we can analyse and understand what is happening in the culture.
Minimise labour: More labour = more staff. Staff cost money!
Maximise computer control: Also to reduce cost of staff. Automation can also reduce human error.
Geometric similarity for scale-up: The reactor dimensions (i.e. height of vessel to diameter of vessel ratio, number of impellers, impeller diameter to vessel diameter etc. ) affect the conditions inside the reactor and thus cell growth. Therefore it is much simpler to scale-up cell expansion by maintaining the same reactor dimensions throughout upstream processing.
Flexible operation: In case of reactor failure, change of cell culture, fermentation modes or reactor types (see Fermentation modes and Types of reactors), a process must be flexible and allow easy changes. One example is to increase the number of reactors you have.
If you have any questions, please leave a comment below and I will get back to you as soon as possible!
In upstream processing there are many different types and sizes of bioreactors. The reactors usually differ based on method of mixing. Each reactor type tends to have it’s own advantages and disadvantages, and some will be more suitable for a certain cell type than others. For example, for an extremely shear sensitive cell it may be better to grow it in a bioreactor that is mixed by bubbles instead of impellers. However, for cells that need a lot of oxygen, a stirred tank reactor would be more suitable since they have good air dispersion. These factors also affect bioreactor size, as explained below.
What does reactor size depend on?
Bioreactor size mainly depends on type of product, product potency and product demand. For example, antibiotics are in high demand as the majority of the population need them, and therefore large vessels are needed. For personalised gene therapy, such as CAR-T cell therapy, a large quantity of the product is not required since the cells are grown only for one individual. Vaccines are also in high demand, but since they are much more potent antibiotics, it does not require as big of a vessel.
Common types of reactors
Stirred Tank Reactor
A stirred tank reactor (STR) is probably the most common type of reactor found in industry. Mixing is achieved via stirring using an impeller, and aeration is done via a sparger (a pipe that pumps in air). Baffles are also used in STRs to reduce vortexing (mini whirlpools) of the fluid.
A stirred tank bioreactor. Mixing is done via stirring with an impeller. Baffles on the edge of the reactor prevent vortex formation.
STRs are generally the most efficient at mixing large volumes of liquid and achieving liquid homogeneity (oxygen concentration, nutrient concentration, are more even throughout fluid). Also, STRs are well-characterised, which has made processes using STRs easily automated. This is why it is the most common reactor in the industry.
However, STRs generally have a higher power consumption (amount of power dissipated into the fluid) than other reactor types. Power consumption is related to the shear stress inflicted on the broth, which can cause damage to cells. Therefore STRs tend to be unsuitable for cells that are shear-sensitive, such as mammalian cells. STRs are best suited for cells that are not shear-sensitive (such as bacterial cells).
Additionally, some cells are anchorage dependent (need to be attached to a substrate in order to proliferate), which is common for mammalian cell lines. These kinds of cells cannot be grown in a traditional STR. However, micro-carriers (basically porous balls that cells can anchor to in a fluid) have been increasingly popular, allowing anchorage-dependent cells to be grown in STRs.
Rocked bed bioreactor
A rocked bed bioreactor uses rocking (moving in a seesaw like pattern) to create mixing in the fluid. It generally consists of a pillow-shaped container attached to a rocking surface (look below). Aeration is achieved via surface aeration – air diffuses from the surface of the fluid into the bulk fluid.
Rocked bed reactor. The cell broth is mixed via rocking. Aeration is achieved via surface aeration.
Rocked bed bioreactors are good for cells that are shear sensitive, since power dissipation tends to be lower. However, surface aeration means that there is lower oxygen transfer than in a STR. Also, a rocked bed system is only effective at low volumes due to difficulty of rocking large volumes and inefficient mixing. Online monitoring and control is also difficult since probes will not be immersed in the fluid all the time (as the fluid is rocked back and forth).
Rocked bed bioreactors are commonly used to scale-up from a flask to a bioreactor, as they can raise cell numbers to higher cell numbers than a flask.
Packed bed bioreactor/Perfusion Culture
Packed bed reactors contain a solid substrate within the vessel for cells to attach to. Mixing and aeration are done by continuous flow of liquid through the vessel. The most common packed bed reactor is the Hollow fibre reactor. The hollow fibre reactor has numerous little pipes spanning the length of the vessel, on which cells can grow on the outside. The fibres contain perforations (holes) to allow liquid to flow through and to increase the surface area.
A hollow fibre bioreactor. Mixing and aeration is achieved by a constant flow of liquid through reactor – waste out, oxygenated feed in.
Hollow fibre reactor diagram (as shown from above). Cells grow around fibres, which have pores to transfer waste into the fibre and feed containing nutrients out of the fibre and to the cells.
Packed bed reactors are good for anchorage-dependent cells as they provide a surface for the cells to attach and proliferate on. They also have better homogeneity than static (not mixed) cultures since there is constant fluid flowing through. As a tank is used, scalability and online monitoring and control is better than in a rocked bed or static culture, especially for anchorage-dependent cells.
However, sampling tends to be difficult and the sample is usually destroyed in the process, due to the complex design. This factor also makes cleaning the reactor very complicated. Cell distribution may also be uneven, which can cause cells to grow differently.
Packed bed reactors tend to be used for expansion of anchorage-dependent cells, though they are getting phased out by micro-carriers.
Shaken cultures
Shaken cultures are cell cultures mixed by shaking. This is done by placing a flask on a shaking system. Aeration is done via surface aeration. Shaken cultures are usually kept inside incubators in order to maintain constant temperature and oxygen levels.
Shaken culture. A flask is placed on top of a shaking platform to mix the contents.
Shaken cultures are well understood and characterised, making them easy to use. This also makes them easy to automate. However, they are not scalable, there is no online monitoring or control and cannot be used for large volumes of cell broth.
Shaken cultures are often used early in the process to grow cell numbers before cell expansion to a rocked bed reactor or small STR.
If you have any questions or want me to explain some other types of reactors, please leave a comment below and I will get onto it as soon as possible!
Bioprocessing Explained 