Downstream processing: Cell disruption methods

Welcome to this next video about downstream processing in the pharmaceutical industry. And in this particular video, what I'm going to talk about is mainly the cell destruction process. So imagine you have your product inside the cell and you need to get it out. What methods can you use for that and what's commonly used in industry? So let's start by giving like a broad overview of what's available. So your ultimate aim is to access the intercellular fluid by opening the cell wall in order to get your product. And you have to imagine that even though you need to break the cell walls, obviously you don't want to damage your product or anything else that's kind of inside there. So it needs to be a very targeted way of how you actually get it out. It's mainly split into two different methods. So we can look at first the mechanical versus the non-mechanical options. And I have to say the mechanical options at the moment do seem to be used more heavily in industry, at least on a big industrial scale, even though the other options are becoming more popular. So in order to make your decision, you need to look at the cell type. So remember the cell wall, for instance, for plants is very different than, for instance, when you have the absence of cell walls in animal cells and the cell type. So for instance, can they withstand high shear stress? So these are all kind of things that you need to think about. And here you can briefly kind of see that overview. So on the non-mechanical side, we'll mainly be looking, I think, like using like a treatment with acid or a base. Enzymes are becoming very popular, and I'll give you a case study of that, or electrical charges. Now, first the mechanical option, that's what we'll talk about first to see and go in a little bit more detail of what options you have here. So you can, here you see a picture of the market. So you can see that the global market for cell lysate and disruption is tremendous. So it's set by 2020 over 4 billion. So here we're mainly talking about the pharmaceutical industry, but as you can see from the graph, there's lots of other industries that can benefit from these methods. So the first option, like blending, similar a little bit to how you have like in a centrifuge, so it uses centrifugal forces in order to break the cell walls. The next step, you can look at beating with beads. So that can be gentle enough in order to keep your cell organelles intact, so in order to keep the product, so you can use like glass or ceramic beads in order to do that. Then we have ultrasonication, where this works with a probe and the disruption of the cell happens via cavitation. So this cavitation, imagine you have these like vapor-filled cavities within a liquid, and once they burst, you literally get like a shockwave coming through. This is shockwave in this case. This is particularly popular for plant and fungal cells, because these are fibrous systems which have very, very tough cell walls. And the most popular method, which will come back to homogenization, that's less suitable for that particular type of cells. So this is a very brief overview. Let's have a little look into the most popular option using a homogenizer and look into a bit more detail on how this works. Now, here you see an example of an industrial homogenizer, and if you look at how this kind of valve works and the pump that comes with it, it hopefully gives you a better idea of how the cell disruption works. So essentially what you're doing, you're forcing a mixture via a very narrow and confined space. And by doing that, you're effectively breaking it. And the kind of forces that come into play, obviously you have the high pressure, but you have things like turbulence, so you might have like turbulent eddies that are forming. You have cavitation, which I mentioned before, but also the liquid shearing becomes important. So you have all of these forces that work onto it, and that's why I imagine that you have something which is relatively large. If you're trying to squeeze through like a small space, and then that way you kind of break it down into smaller parts. And so you need like a pump and a valve in order for this to work, and this can actually, later I'll show you, you can also do this continuously. So you can do this on a very large scale. And while in this video specifically on cell disruption, you can also use this system for instance with food and the beverage industry, because it also works for emulsions and suspensions. So there's lots of applications in other areas as well. And then, well, we moved away now from the mechanical methods, which I did say were the more popular option, and then let's look at some of the non-mechanical methods. So in general, what the advantage of this is, is that you use milder conditions. But bearing in mind, except for the physical methods, if you use a chemical and enzymatic method, you require additional compounds, which you might need to filter or purify out later. Obviously, it comes with additional costs as well, but also the purification of these compounds can be a bit tricky. Now, the physical methods that you can work with, like freeze-thaw, osmosis, you can have a thermolysis or electric shocks. The more popular methods are like the chemical methods, where you can use some organic solvents, or you can use charged compounds like detergents. And here you can kind of see an example of how this works. So a compound like EDTA chelates cations, and these ions are very important in keeping your cells stable. So what you will see, it destroys your cell membrane because it draws away some important cations that are crucial to the stability of the cell. And by doing that, you can slowly release the intracellular components that you're after. Now, enzymatic methods, this will come back into one of the case studies that I will show you. We have a wide range of enzymes available for this respect, and this has some advantages, but you will also see this often tends to be quite like a costly option. So here, let's come back to example of case study number one. And if you want to know more about it, you can go to the link below. And this was where they were looking at enzymatic cell disruption of microalgae. Now, these microalgae, they can be used for many different things. So you can use them as a kind of host, where they can produce different proteins or different lipids. There's also talk about using them in, for instance, biodiesel. So you will see that more and more, like that is becoming a bigger market for microalgae. In this particular paper, they were interested in atrius enzymatic-assisted extraction to retrieve the lipids, because compared to like the mechanical methods, it's a very fast process, but it is selective. So some of the other options, where you're just mechanically kind of taking it out, are less selective. Another big advantage of microalgae is all about sustainability as well, is that you're working on the relatively mild conditions. So relatively low temperature, relatively neutral pH range. And that's also a big advantage. In most of the cases, you don't need additional drying steps. So energy-wise, it's also efficient. But you will see here, like in the next slide, there's a picture of it. We still needed to dissolve some of the lipids in some solvents, so you would still have to have some drying. In most of the cases, though, the costs are the limitations for this. And this is also addressed in this paper. And that's one of the current drawbacks, because you need it in relatively large quantities. And also, obviously, you need to optimize it fully to the system. So when you're working with the enzyme, you really need to screen for a range of enzymes. And in this case, they found out the lysine, which was very selective, because it only targeted proline residues within the cell wall. So it would only work on the cell wall itself. But in order to kind of work this out, you need to go through a whole library of enzymes. And not just that, you need to optimize all the steps, so the buffer that you're working on, the pH, the temperature. So this can be a very time-consuming process, whereas when you do the mechanical option, it's a more general process. And you will see you don't have less optimization steps in there. But then let's have a look at what it actually looks like. So here you can get an idea of how the cell wall works. So there is a control sample on the left. And you can see that the cell is nicely intact there. There's a couple of components in there which are labeled. But if you were treated with autolysin on the right, you could see that the cell was kind of burst open. So that was after two hours, so it's relatively fast. What you can see a little bit here is that some of the lipids were still sticking to the cells. So in this case, they really needed desolvents in order to dissolve them afterwards. So that's why you will often, when you look at these cell lysis, and you will also see this in the next example, you will often see there's a combination of different methods in order to make sure that the process is more effective. But enzymatic options will relatively become more popular because after doing more research on it, we'll know more about the optimal conditions and more about certain enzymes that we can use. And as they will become more popular, hopefully they will also help to reduce the costs because they do have a very big sustainability advantage. Now, in the second example, I mentioned before that these high-pressure homogenizers are the ones that are used in a very large industrial scale. And a big advantage of those is that you can also do this continuously if you do the cell lysis in a single pass. And this wasn't really possible with the old models of homogenizers, but definitely it is possible now, and that really reduces the cost, which is why they are by far the more popular option. Now, the flow rate that you can achieve of 50 liters per hour is way too high for small pilot-scale reactions. So you will see on smaller-scale reactions, you do tend to use other things like the ultrasonication or maybe enzymatic or chemical methods. And one of the papers here that they mentioned, and I mentioned this before as well, is that you often can combine some of these techniques. And you can get really, really high efficiency when you use these homogenizers. So here they combined a kind of mechanical option with a detergent, so a chemical option, and they reached over 97% efficiency. So very, very high efficiency in terms of disrupting your cells. There are some things that you will need to consider here. So your products that you're working with, you are imposing a high shear rate. So it should be high enough in order to break the cell walls, because what you don't want to do is damage the product in the process. So that's something that you really need to consider what you're working with. I also mentioned before that if you're working with plant cells, you often tend to use other systems. So if you have more, let's say, tough fibrous cell walls of a plant, if you want to use this with a homogenizer, you are often working with frozen systems where they become a bit more flaky, so they become a bit more brittle and easier to break. However, particularly when you're working with animal cell walls, due to the absence of the cell walls that you have, they are much easier. So you will see definitely that these high-pressure homogenizers are the common standard. Now, this was a very short video, so let's have a brief summary. So the cell disruption is really an essential process and downstream process. So you really need this in order to retrieve and extract your products in most of the cases, because your products are often produced within the cells and not outside of it. There are two common routes, so I discussed the mechanical versus non-mechanical option and discussed what the common options are. And this route really depends on your cell wall and type, so you need to consider that. And then furthermore, there are also things like always the cost is important, the yield that you want to achieve, because some options actually give a higher yield, but also consider the stability of your products when you kind of select the right option for you. So this is a very brief summary. We have a lot more videos on downstream processing. For instance, if you look at a more practical example of how you would use this for antibodies, then do have a look at this playlist. Thanks for watching.