In the bioprocess field, we call upstream processing to all the operations that need to be prepared before the fermentation starts. Usually, these operations include the preparation of the medium, the sterilization of the raw materials and the inoculum preparation (seed-train).
Keep reading to get a better understanding of these different points.
Preparation and Storage of Solutions
The main example that should come to our mind when thinking about these kinds of solutions is the medium preparation. In this case, liquid and solid components are filled in a tank and mixed by agitation. Normally, when a homogeneous solution has been achieved, the mixture needs to be sterilized. These can be done either by filtration or autoclaving.
Each process or biotech plant should take into consideration its own scenario before deciding which way would be the best. In general terms, it is complicated to autoclave a big amount of medium.
If possible, it is better to prepare solutions with a higher concentration to keep the working volume in that step low. It is easier to work with small tanks and small volumes, and additionally quicker to pump it to the final recipient. The solution is hereinafter diluted in the bioreactor to achieve the target concentration.
Another important consideration when sterilizing with head is to separate carbon and nitrogen sources to avoid the formation of Maillard or non-enzymatic browning.
Additionally, it is important to define the desired volumes to work with because it will determine the concentration that is needed in the different solutions that are prepared. It is worth mentioning, if we want to avoid undesired precipitation in the media culture, we ought to define the order in which the components are added.
Finally, if we are going to store these solutions for a period of time, we must do a preservation test to ensure that the quality of the prepared solution is maintained until and while we use it. It is advisable to test it for a longer period of time than the one it is planned; to be secure it maintains the desired quality.
Sterilization of Input Materials
Input materials of a bioprocess needs to be sterilized to avoid contaminations and ensure that only the desired microorganism is growing in the culture.
This is a key point in all the bioprocesses and under my personal experience it could be one of the main causes of batch KO’s when producing. As we are going to see, this process is based in a probabilistic phenomenon where one assumes sterile condition when the possibilities of survival of certain microorganisms are close to 0.
We can find two types of sterilization: by heat or by filtration. Let us see it:
> Heat Sterilisation
When sterilising by heat, dead kinetics can be expressed as ln(Nt/N0)=-kt where N is the number of viable microorganisms and k is the dead constant velocity.
If we look into the equation, we can assume that to achieve Nt = 0, infinite time is needed and that for long times Nt < 1; which has no sense, because it would mean a fraction of a microorganism. However, we should interpret it like a probability of microbial survival.
Therefore, when we talk about autoclaving something, we can not assure that there would be 0 microorganism alive. In fact, as was said before, it can be assumed that something has been sterilised when the probability that an adventitious agent is there is less than 10-3. That means that Nt is 0.001 and that there is 1/1000 probability that a microorganism survives the sterilization process.
The kinetic constant k that was defined in the last equation depends on the temperature and can be expressed by the Arrhenius equation. K=Ae^(-E/(RT)). Where: E: activation energy, A: pre-exponencial factor, R: gas constant and T: absolut temperature.
Therefore, the equation now would be ln(N0/Nt)= Ae^(-E/(RT))·t. We can develop this equation deeper in another post.
However, the main point here is that we can achieve a certain degree of sterilisation playing with two key parameters: the temperature and the exposure time. This mean, we can use high temperatures in small amounts of time or lower temperatures for long periods (this is called pasteurisation).
Normally, this temperature is achieved by indirect heat transfer between the steam inside the reactor jacket and the culture inside the reactor. Though, it can be achieved too by injecting steam directly into the culture. However, this method inevitably leads to dilution of the culture resulting from steam condensation. It is not a major inconvenient given that it can be considered beforehand.
> Filtration Sterilization
In this case the media is pumped through a filter with a final porous of 0,22 µm. It is the best solution when working with thermolabile culture media or with larger volumes; where it would be difficult and expensive to ensure heat sterilization in the whole reactor.
Normally, a pre-filter of 0,65 µm or 0,8-0.45 µm is added to ensure that the sterilizing filter (0,22 µm) does not foul quickly. This will facilitate the procedure and extend the useful life of the filters.
Also, it is important to sterilise the gaseous streams to ensure sterility inside the bioreactor is not broken. A filter with 0,2 µm will be used too.
This is a crucial phase, where enough active cells should be provided to the production fermenter to properly start the fermentation.
Normally, seed-trains starts from a small amount of frozen culture of the cell line that will be used in the bioprocess. One vial should provide enough inoculum for the starter culture of the seed train for each batch. Seed trains are grown under conditions that enable high cell densities of actively growing cells in short times.
> Seed train for bacteria culture
In general, the first steps of a seed train for a bacteria culture are developed culturing inside Erlenmeyers that become bigger in every step. For example, if an Erlenmeyer of 250 mL it is used for the 1st step; then, it is common to use one of 500mL or 1L in the next step.
Then, small bioreactors are used to achieve enough biomass. For example, an Erlenmeyer could be used to inoculate a benchtop reactor (5L) that once has grown could inoculate a bigger bioreactor, for example one of 100 – 200L.
This is repeated until enough biomass is available to inoculate the production reactor.
> Seed train for cell culture
The typical sequence of an animal cell seed train is quite different from the bacteria. It will involve T flask > roller bottle > disposable bag bioreactor > first seed reactor > second seed reactor > production fermenter.
The medium’s composition of the seed train can be quite different from the one used in the production reactor to maximize cell growth. However, it is highly recommended to adapt cells to the final medium in the last step of the seed train.