Hello, my name is James Lawrence from the Department of Biochemical Engineering at University College London and I'll be talking through this video on recovery and purification of small molecules. So all products derived from biological processes will need to be recovered and purified before they can be sold or used in the manufacture of a different product. The design of the process we use to do this recovery and purification will depend on the type of product, the product's location, and the nature of the contaminants present. For the purpose of this course, we've broken down the type of product into two categories, small molecules and large molecules. By small molecule, we mean a molecule of with a low molecular weight. So for example, ethanol or insulin. And by large molecules, we mean molecules with a high molecular weight. So for example, enzymes or antibodies. In this video we will discuss the recovery and purification of small molecules, and we'll move onto large molecules in the next video. The location of the products will also impact the recovery and purification process. When we say location what we mean is whether it is retained within the cell that has produced it, i.e., whether it's intracellular. Or if it is secreted into liquid surrounding the cells, so if it is extracellular. All recovery and purification processes will be made up of a series of separation of steps. Each of which will exploit some difference between the desired products and the other compounds that are present which we term the contaminants. This difference can be any one of density, size, charge, hydrophobicity (which is the degree to which the products or contaminate repels water), the affinity for a particular molecule, or a combination of several of these differences. It's worth noting that contaminants in a cell based or biocatalyst based process will be different. In a cell based process we will need to remove the cells and possibly cellular debris as well as the culture fluid components all from our product. We might also need to extract the products from the cells before we can start the purification process. If the process is biocatalyst based, we will need to remove the enzymes, if they haven't been immobilized, as well as any unreactive substrates or any cofactors required. It's also worth bearing in mind that most of our processes will take place in water, and our product concentration will typically be very low. One of the first priorities of any recovery and purification process then is going to be to remove as much of this water as possible, as soon as possible to minimize the size and cost of operation of the latter stages of the process. Recovery is the name given to the initial stages of the separation process in which we will separate out and concentrate our product and remove the bulk of the contaminants and water from it. We typically use filtration or centrifugation for this purpose, as they can handle large particulates like cells. Filtration is driven by a difference in size of particles, while centrifugation is driven by a difference in density. So in filtration we pass the fluid through a membrane with small pores of a defined size. Only liquids or particles smaller than these pores will be allowed to pass through the membrane and into the permeate. And larger particles will be held back in the membrane in what we call the retentate. Filtration is quite a useful step for separating cells from culture fluids. As the bulk of the culture fluid will be allowed to pass through the membrane, most of the cells themselves will be retained within the retentate. The filtration units can typically be operated in two modes, by the normal flow or tangential flow. In normal flow mode, the fluid will be driven directly into the surface of the membrane, whilst in tangential flow mode, the fluid is driven over the surface of the membrane, flowing in parallel to it. In both cases, there is a pressure gradient from the retentate side to the permeate side, which drives the fluid and small particles through the pores. However, operating in tangential flow can be advantageous, as the flow of fluid over the surface can prevent larger particles from accumulating on the surface and blocking transmission through the membrane. Centrifugation is the other main choice for the recovery of cells and is very commonly used where large volumes of fluid needs to be processed. As centrifuges are typically capable of much higher throughputs, so they can process more fluid per hour. There are two main types of centrifuge, the tubular bowl centrifuge and the disc-stack centrifuge, but both operate on the same principle. this being the use of centrifugal force to displace particles of a certain density. So the centrifuge has a rapidly rotating chamber into which our fluid flows. The rotation of this chamber subjects the fluid and the particles within it to force many times that of gravity. This force causes particles to move away from the center of the chamber. Most of the fluid itself, is allowed to flow through relatively unimpeded. The denser the particles are, the more rapidly they'll settle out of the fluid toward the walls of the chamber. In the case of the tubular bowl centrifuge, they will accumulate on the side of the bowl. And in the case of the disc-stack centrifuge the particles will accumulate towards the corners of the centrifuge. We can adjust the rotational speed and the flow rate through the centrifuges to obtain particles of a particular density, usually our cells, whilst allowing the rest of the fluids and less denser particles to pass through. If our product is intracellular, we will need to extract it from our cells by breaking them open after the recovery stage. This process is called lysis and it can be performed using chemicals, to dissolve the cell membrane, or high temperatures or pressures to destabilize the membrane. In industrial biotechnology, we typically use pressure to lyse the cells, as it is the most efficient method and doesn't require the addition of further chemicals. We use a device called a homogenizer which uses pressure to break the cells open by forcing them through a partially closed valve and into the so called impact ring. The constricted space of the valve accelerates the cells up to a high velocity so that when they collide with the impact ring the cell membranes will break open. This releases our product into the surrounding fluids, but it will also release cellular debris along with it. So on further purification using a filter or centrifuge is usually required to remove the bulk of this debris. So the recovery stage of an industrial biotechnology process typically has a very similar structure, regardless of the product that we are trying to produce. But the purification stage will vary quite widely for different small molecular products, as there are quite a lot of unit operations to choose from. So rather than going through all of these steps in this video, we'll look at a couple of case studies for the manufacturing of two different products. The first being L-glutamate and the second being bioethanol. So L-glutamate or glutamic acid is an extra-cellular product which is used as a precursor for pharmaceutical compounds and also for flavourings like MSG. It's produced by fermentation using a few different bacteria. And after the fermentation and recovery stages are complete, there are two main steps which are required for the purification stage. These are crystallization and filtration. So crystallization exploits the varying solubility of compounds in different conditions to cause them to form crystals, precipitating them out of solution as solids. The concentration of the product that we are trying to crystallize, as well as the pH of the solution, and the temperature of the solution, can all be used to affect the product's solubility. Since different compounds will have different solubilities under different conditions, we can choose our parameters such that only our L-glutamate molecules will crystallize. The crystallization process consist of two main steps. First is nucleation, where the crystal starts to form nuclei over few molecules. And then we have crystal growth, where further molecules join the structured nuclei and form larger and larger crystals. Once the crystallization is complete, the suspension of our glutamate crystals can be filtered to separate them out from the contaminants and the liquid. The crystallisation is not always perfect and sometimes we will end up with other contaminating molecules in our glutamate crystals. But if this happens, we can simply redissolve the crystals and repeat the crystallisation process until we reach our desired level of purity. Bioethanol which is a somewhat lower value product than glutamate, is also an extracellular product which is typically produced by the fermentation of yeast or E.coli strains. The main challenge of bioethanol production is that the ethanol is toxic to the cells and will cause them to die if the concentration of the ethanol within the solution is too high. So to prevent this we need to remove the ethanol as it is generated. And we can do this in one of two ways, either by continuous mode fermentation, as we discussed in the previous video on cell culture, or by high temperature fermentation, where the cells are grown at about 50 to 60 degrees C, which causes the ethanol to evaporate out of solution as it is formed. If we use continuous mode fermentation, then the liquid leaving the fermenter will need to go through a recovery step to extract the cells before it can be purified. If instead we use the high temperature fermentation method, then we can simply condense the ethanol vapor from the fermenter and skip straight to the purification steps. So the first step of our ethanol purification will be distillation, which exploits the various boiling points of the different compounds within the solution to effect separation. So a gradient of different temperatures is applied over the length of the column, with lower temperatures at the bottom, and higher temperatures at the top. The fluid containing ethanol is put into the middle of the column and the ethanol itself will evaporate and rise to the top of the column. Whilst the water and other compounds which have a lower boiling point should condense and drop to the bottom. Additional vapor can be extracted from the column but we can only ever reach about 95% purity with normal distillation. Now this is because a solution of 95% ethanol and water actually forms a complex that has a lowering boiling point then a solution of pure, 100%, ethanol. So now matter how many times you put this complex of 95% ethanol in water through our distillation pump we'll never be able to separate out that remaining 5% of water. So if you want to get to a higher purity of ethanol, we need to use techniques called molecular sieving or reverse osmosis. Both of these techniques utilise materials with nano-scopically porous structures which are so small that only water molecules can pass into or through them. So molecular sieves are particles made up of these nanoscopic structures which could be added to the ethanol and water solution that comes out of our distillation column. The water molecules will diffuse out of the solution and into the pores of the sieve particles, and the particles can then be removed leaving pure ethanol behind. Reverse osmosis, on the other hand, is essentially a filtration process which uses membranes with nanoscopic pores. In this case, the fluid is flowed into the membrane, but only water molecules are able to pass through. So we'll be left with our solution of pure ethanol.
