 As we discussed in our previous module that what are the different mechanisms involved in filter sterilization or the filtration, but in particular in this module we will talk about the use of the filtration for the sterilization of the fermentation medium. So, as we discussed in our previous modules that when we are dealing with the sterilization of the medium using the heat then there is the chance of the nutrient loss. So, when the medium is or a media is very sensitive to the heat then there is the only choice the use of the filtration. So, especially when we are dealing with the medium for animal cell culture so that cannot be sterilized by the steam because that contain a very heat liable proteins. So, thus the filtration is the method of choice and the fixed pore or absolute filtration is the better system to use. So, an ideal filtration system for the sterilization of the animal cell culture must fulfill the following criteria. So, these are the criteria that should be fulfilled for the filtration process. Number one that is the filter medium must be free of fungal, bacterial and mycoplasma contaminations because when we filter the medium and we should assure that that should be properly removed either that will be fungal, bacterial or mycoplasma. So, if any medium is passed through the filter then we should assure our self that should be properly removed from these agents. The second there should be a minimal adsorption of protein to the filter surface. So, if we are dealing with the electrostatic interaction in some cases some proteins can attach to that filter. So, if we are using the filtration process then we should very careful about that there should be as minimum as the adsorption of the protein to that filter surface. As concerned the third requirement the filtered medium should be free of viruses. As concerned the fourth the filtered medium should be free of the bacterial endotoxins. This is very critical because if we say that the medium is filtered and that is free from all those fungal, bacterial and mycoplasmic organisms are the contaminate contaminants. But sometimes when there is an old and the media remain for a long time then there is a growth and during the result of the growth in that medium what happened those microorganisms secrete some endotoxins into that medium. So as concerned the filtration mechanism that cannot be removed those endotoxins. So we should assure that if we are using the filtration we should avoid such the production of such a bacterial endotoxin into that medium because we cannot remove such endotoxins which can alter which can if have a negative effect on our fermentation process. So as concerned there are the several filter manufacturers now apply the absolute filtration system for the sterilization of the animal cell culture medium. So such system consists of the membrane cartridges which are fitted into the stainless steel and those filters are steam sterilizable modules. So the membrane for the medium sterilization are constructed from the steam sterilizable hydrophilic material and are treated to produce a filtrate of particular quality. So for example if the minimal protein adsorption is a major criteria then a specially coated filter membrane is used. So it would be very difficult to construct a single filtration membrane which would fulfill all four criteria as we discussed in our previous slide. So thus a series of the filter are used to achieve the desired result. As you can see in the next slide in this slide you can see there is a series of filters. So you can see here this is the bulk pooling tank so the vent here because if there is a pressure and vent and then there is a pressure so filter one, filter two, filter three and filter four. So as concerned all these four filters so you can see what is the function of the first filter. So the pore size in first filter will be the 5 micron meter which absolute rated pre-filter for the remover of coarse participates and then the second filter having the size and the pore size of 0.5 micron meter that absolute rated for a pre-filter for bulk bioburden removal just to increase the life of the third and fourth. As concerned the third filter that having the pore size of 0.1 micron that absolute rated the single layer pre-filter for further bioburden and endotoxin removal. So as concerned the fourth filter that having the pore size of 0.1 micron meter that is also absolute rated double layer final filter. In this you can see that the single layer but in the fourth filter that is the double layer for final filter and absolute sterility that is used for the micro plasma removal and further if there is any minor and the micro quantity of the endotoxin. So as concerned in other you can see here that if here is the fermenter and then if we are dealing with the so the filter 1 and filter 2 in case of the represent the filtration shift of the removal of the cell and the cell depress from the animal cell culture fermentation. So in this case the first filter having the pore size of 1 micron just to rated the pre-filter for bulk cell and the cell depress as concerned the second that is the 0.2 micron. So that is the single layer bio inert filters for final bioburden removal. So in this case there is a series of the filter that is used for the proper sterilization. So the pre-filters are used as we have seen in our previous figures that the pre-filters are mostly used for the removal of the coarse particles especially they are the bigger particles. If we are not using the pre-filters then there is a chance of the blockage of the filters. So just to check just to avoid the blockage of the fine filters and just to increase the life of the filters then there is the need of the pre-filters. So we have to line up all those filters just decreasing the pore size from the first to the next series of the ferment for the filters. So if we are using the filters in a series then we can have the surety of proper sterilization.