Hello, my name is James Lawrence from the Department of Biomedical Engineering at UCL, and I'll be talking you through this video on recovery and purification of large molecules. So in the last video, we discussed the fact that all of the products derived from biological processes will need to be recovered and purified before they can be sold or used in manufacture of a different product. The design of the process that we used to do this the recovery and purification will depend on the type of product, product location and the nature of the contaminants present. As a reminder for the purpose of this course, we have broken down the type of product into two categories, small molecules and large molecules. When we use the term large molecules, we mean molecules with a high molecular weight. For example therapeutic proteins, monoclonal antibodies or enzymes. The production of large molecules will be done almost exclusively by living cells, since the assembly of these molecules requires processes that could not be carried out by individual enzymes or even a series of enzymatic steps. The design of the process by which we recover a large amount of products is therefore going to be affected primarily by the location of the product within the cell, i.e. whether it is made inside the cell, it's an intercellular product, or whether it is going to be secreted out of the cell, so it's an extracellular product. And that, in turn, is going to be defined by the type of cell producing it and the size of the molecule itself. As an additional complication, a loss of these molecular problems tend to be biopharmaceuticals, meaning that the products need to be purified sufficiently, so that they could be administered to a patient without causing harm to the patient. Usually this means purity in excess of 99.95%, and this is a legal requirement that must be required for any pharmaceutical. If we have an intracellular product, as would be the case if we had for example an E.coli cell making a large molecule like an antibody fragment, then we would need to recover the cells and homogenize them to get out our product out, as we described in the previous video. If we had an extracellular product, we would not require any homogenization , simply the removal of the cells from our products using a standard solid- liquid separation method, such as filtration or centrifugation, also described in the last video. The last process that we go through is most likely to be used because it typically has lower cost associated with it and is capable of having higher throughputs. The choice of cell type in our process is often influenced by the complexity of the product, and how easy it's going to be to subsequently purify it. So for example in mammalian cell lines such as a Chinese hamster ovary cells, we'll typically be able to secrete a large product that a microbial cell would have to keep intracellularly as it couldn't express that through a cell membrane. This means that using Chinese hamster ovary cells to produce a large molecular product will reduce the number of steps that we're required to purified. However, there's going to be a tradeoff, because as a rule of thumb, the simpler the cell type we use, the higher the cell mass we can achieve, and therefore the more product we are able to make overall. A potential extra step in the purification process for large molecules would only occur in the specific case of a bacterial cell overproducing our molecule of choice. This overproduction would lead to misfolding and aggregation of the proteins within the cells, creating what are called inclusion bodies, which are small particles of misfolded proteins, large particular proteins which tend to be about half a micron in size. They are small robust particles, which are very hard to break apart. So they are actually relatively easy to separate from the cells and other contaminants present. We do this by breaking the cells and the resulting cell debris into tiny pieces, and then centrifuging as the resulting suspension to separate out the more dense inclusion bodies from the very fine broken down debris and other contaminants. After the inclusion bodies have been separated out, but before any further purification can occur, the bodies must be separated out into individual molecules by the addition of a chaotrope. There's a chemical that is used to unfold proteins. The advantage of making inclusion bodies is that you can often get more products per cell. And actually it simplifies the further purification steps, provided that you can actually unfold and refold your products back into its natural active form. So, having extracted our products from our cells, if that's necessary, our next step would probably be chromatography. So chromatography involves the passing of liquids, known as the mobile phase, through columns of packed with, porous beads, which are known as the stationary phase. In doing so, we can use the beades to effect a separation of the molecules passing between them in the liquid, on the basis either of the size of our molecules, the charge on our molecules, the hydrophobicity or their affinity for a particular ligand. Chromatography units can be operated in one of two modes to effect the purification. The first is 'bind and elute', where we bind the products within the column, let everything else flow through, and then release the products again, leaving it as a pure stream on its own. And the second mode is a flow-through, where we bind specific impurities within our product stream and let the product flow through itself. So size exclusion chromatography, as the name suggests, separates our molecules out based on their size. The smaller molecules are absorbed into the pores of the beads in the stationary phase and have to move through these beads, whilst the larger molecules can move only through the gaps between the beads and column. This means that the smaller molecules have to travel through the column much more slowly. They are inhibited by the fact that they have to travel through these small pores within the beads themselves, and they will actually come out of the column known as eluting, later on than larger molecules. So there's no attraction to the beads themselves, there's no attraction between either contaminants or products in the stationary phase. This type of chromatography is always operated in flow through mode. Ion-exchange chromatography separates based on charge, which could be positive or negative charge known as anion or cation respectively. Put another way, cation-exchange chromatography retains positively charged molecules, because the stationary phase has a negatively charged, functional group, on its surface. Conversely, anion-exchange chromatography, retains negatively charged molecules, using positively charged, functional groups on the surface of the stationary phase. So, if we were using cation-exchange, in our process, and our product was positively charged, we would be operating a 'bind and elite' mode, as we would be capturing our products on the column, whilst our contaminants would be allowed to flow through. If the product was negatively charged, we would be operating in flow through mode. As the product would be repelled by negatively charged stationary phase, whilst any positively charged contaminants would be captured by the column. The opposite would be true if we were using anion exchange instead. Finally, the other two types of chromatography, hydrophobic interaction chromatography and affinity chromatography both work in quite similar manner. In this case, the stationary phase is designed specifically to bind molecules based on either hydrophobicity or the affinity of molecule for a specific ligand, which should be held on the stationary phase. These types of columns tend to be used as the final steps in our purification process, As they allow the capture of very specific molecules, so can reach very high purities, but they also tend to be very expensive. So we generally want to remove as much of the contaminating material as possible before we get to one of these steps. So, lastly just presenting an overview of all of the recovery and purification methods that we've covered in detail in the last two videos, and the different properties by which they exploit to effect a separation. You noticed, that we haven't included homogenization in this list, as it's not really a separation step, but instead a pre-treatment step, used to break open cells and access intacellular products. As a final note, it's worth mentioning that, if the large molecular product was made by a mammalian cell line, then there would need to be both a viral inactivation and a viral filtration step within the purification chamber after any chromatography steps. Again, this would be a legal requirement to ensure that any pharmaceutical products we made using ourselves, will be safe for use for humans, or indeed, for animals.
