Hello everyone and welcome. My name is Suzanne Bellmore and I will serve as your moderator today. Thank you for joining us for today's webinar Process development, intensification for ultra filtration, diet filtration and Viral vectors. As your moderator, it is my role to ensure that we make the most of your time with us. I am here today with Anand Alambath and Rosario Servalier. Anand is a development engineer and the Applications Technical lead for to Denial flow filtration product development projects with a focus on downstream purification of vial vectors. Anand holds an Ms. and PhD in Chemical Engineering from Missouri University of Science and Technology, Rolo. Rosario is an applications engineer in filtration R&D. Since joining in June 2021, he has supported technology and product development projects including Pelican capsule with 100 and 300 kDa Ultra Cell membrane and Pelican XL50 cassettes. His recent focus applies modeling and simulation to increase efficiency of product and technology development. Azario earned his PhD in Mechanical engineering from the University of Arkansas with a focus in computational material science and global change. Before I turn things over to our presenters, I'd like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets you can use. There you will find our reaction button. Indicated by the thumbs up emoji that allows you to give immediate feedback on the presentations, topics, or anything that stands out. All the widgets are resizable and movable, so feel free to move them around to get the most out of your desktop space. You can expand your slide area or maximize it to full screen by clicking on the arrows in the top right corner. If you have any questions during the webinar, you can submit them through the Q&A widget. We will try to answer these during the webinar, but if a more detailed answer is needed or if we run out of time, it will be answered later via e-mail. Please know we do capture all questions. You will also have an opportunity to participate in a couple of quick poll questions throughout the session. I encourage you to take part in these surveys. If you're watching this webinar on demand, you can still submit poll responses and questions via the QA widget. The webinar is being streamed through your computer, so there is no dial in number for the best quality audio. Please make sure your computer speakers or headset are turned on and the volume is up so you can hear the presenters. An on demand version of the webinar will be available after and can be accessed using the same link that was sent to you earlier. So that's it from my side. It's my pleasure to turn things over to Anan and Rosario. All right. Thank you, Suzanne, for the introduction. Hello, everyone. Welcome to today's webinar on process Development and intensification for ultra filtration and difiltration of viral vectors. The topics which we would like to cover today will begin with an introduction to tangential flow filtration. Will highlight the importance of concentration and dye filtration in the downstream process. For viral vectors, we'll discuss the different operating strategies. For tangential flow filtration, we'll cover the scaling performance of single use TFF filter. Then Rosario will discuss the TFF module comparison of the spiral cassette and hollow fiber, and finally. We'll conclude with the key takeaways. Before we start, we do have some poll questions. As Susan mentioned, we would request all the participants or the audience to participate in the poll question. The first question is what vectors are currently being targeted in your process? Is it the adeno associated virus? A AV? Are you working on lenti virus LV or the adenovirus AV? Or is it something nonviral like the lipid nanoparticles? Or is it something different which is not in the list? Feel free to choose multiple options if you're working on multiple different vectors. Thanks everyone for your participation. For today's webinar we will be discussing only the Adeno associated virus but it is pretty much the data is pretty much applicable to all the different application within this space of seven gene therapy. I'll start with an introduction to tangential flow filtration. This is the general downstream process for AAV and plenty virus. As you can see there are two TFF step, one before the chromatography and one after the chromatography, one before the chromatography we typically call it as TFF, one it is used to. Concentrate to reduce the loading time and enable buffer exchange for chromatography. It can also do some partial purification like removing benzonase endonucleases that are used to digest the DNA. It can also be used to remove other proteins and DNA fragments. D FF2 is typically used to achieve the final concentration and exchange into the formulation buffer. For both these steps, 100 or 300 kDa membrane cut off is used. So the goal of the TFF process is to retain the molecule of interest, in this case the viral or the nonviral particles and allow the impurities to pass through the permeate the key parameters that are critical for the tangential flow filtration process. The first one is the membrane selection which includes the choice of membrane material and pore size. For these applications we are using 100 or 300 kDa membrane cut off and the membrane material that we used as a regenerated cellulose ultra cell membrane. The second one is the trans membrane pressure. It is basically the driving force. It is the difference in the pressure between the feed side and the permeate side. And the final one is the cross flow flux, which is the average flow rate across the feed channel. This this is particularly important because this this is the one that reduces the falling by preventing the accumulation of particles. So there are two ways you can control your tangential flow filtration process. The first one is the trans TMP control, the trans membrane pressure control. The second one is the permeate control. The TMP control is achieved using one feet pump where the flux and the TMP is controlled by adjusting the retentate backpressure valve. The TMP control is generally used for tighter UF membrane applications like the 30 kDa for antibody retention in contrast. For the permeate control, you have a secondary permeate pump to control the permeate flux. You can as an alternate, you can also have a valve on the permeate side to control the flux. Permeate control is typically used for more open membranes like the microfiltration applications. Where because your membrane is really open, the fluxes can be high and can lead to premature falling of the filter. So you need a secondary pump or a valve to control the flux on the permeate side. For viral vector applications membrane cut off 100 and 300 kDa is used and it lies between the TMP control and the permeate control before we run the. Concentration and difiltration. We need to characterize the flux and the transmembrane pressure, which means you have to identify what are the ideal operating conditions so that you can complete the TMP control. You can complete the concentration and difiltration in stable conditions. The way you characterize the TMP control and permit control are different. For TMP control, you perform a TMP excursion where you measure flux at one psi. TMP increments by adjusting the written date valve. The goal is to identify the initial TMP, which we defined it as the .1 point before the plateau. So the plateau is again called the. Is also called the pressure independent region. It is defined as three consecutive points of increasing TMP where the difference in flux is less than 10%. So once we identify the plateau, one point with the point before the plateau is our initial operating flux for the concentration. In contrast, for the permeate control we perform a flux excursion where we. Increase the flux by increasing the speed of the pump and as we increase the flux we keep monitoring the TMP. At each flux level. We're trying to identify the point where the TMP rises rapidly. So you see there's a sudden rise in TMP in short interval of time and that's called the critical flux. Once we identify the critical flux, the rule of thumb is to operate at 50 to 75% of critical flux for your initial operating flux during your concentration. So now what I'm doing is I'm overlaying both the TMP control and the permit control flux and TMP profile in one graph. And try to establish the difference in the two control modes. So for TMP control there is a minimum trans membrane pressure that is needed to generate the cross flow flux. So in this case we're trying to run the filter at 5 liters per meter square per minute and you need a certain minimum trans membrane pressure to generate that cross flow flux. So permit control. Since you're restricting on the permeate side, you are able to operate the process or run the process at lower TMP. When you compare the curves of TMP control and permit control, you see that the flux profiles are very similar, especially the critical flux in the permeate control. Lies in the plateau region of the TMP control. As you can see, the minimum TMP in TMP control corresponds to 80% of the critical flux and for permit control you can very well run it at any flux level. So you can operate at the stable 50% of the critical flux during your concentration now since both control modes can operate below the critical flux. Which is preferred for our applications. So that's the first part of my talk where I'll be comparing the TMP control and the permit control before we get into the comparison. We have our second whole question. The question is what are the biggest challenges you face within TFF processing step? So is it a the cost of the filter itself or is it the? Impurity reduction. Is impurity clearance a target in your TFF step? If yes, do you face any challenges? Option three is low yields. Are you facing low recovery of viral vectors during your TFF step? Option D is linear scalability of device sizes. Do you see issues when you switch scale in performance? Option E as long processing times as your filter experience low flux, consequently more higher processing time. Or is it the operator safety? Does your system, TFs system or filter? Is it? Is it? Is it more prone to open processing and more prone to exposure to your operators? Or finally the long installation and cleaning procedures that are very. Comment in your in the TFF step. Again thanks everyone for your participation. These are very valuable insights so that we understand the pain points you're facing in TFF and and we can provide specific solutions to it. Then let's. Head on to the TMP control and Permit control comparison. Again, the goal is to do a head on head comparison of two control modes. The processing step is involves 10X concentration and five DB dye filtration. The way we achieve the 10X concentration is through two concentration step. First we concentrate to 4X concentration. And then switch to dye filtration and perform 5 DV dye filtration at constant volume and when finally concentrate 2.5 X the dye filtered solution taking it to a total of 10 X concentration. The operating conditions we maintain the cross flow flux at 5 liters per meter square per minute. The key performance parameters which we are monitoring the first one is the average flux and processing time. So for TFF one before the chromatography, speed is very important. The goal is to reduce the loading time in your chromatography step, so you want to run the TFF as fast as possible. While we run it fast, we also want to keep the pressures very stable. Internally, anything less than 15 psi TMP was considered within the processing limits. The next one is the virus eel. We want to have highest recovery as possible from the TFF one step and finally impurity clearance. Again. Impurities like endonucleases that helps to digest the DNA can be easily cleared using the 100 and 300 kDa membrane cut off during your TFF one step. For these experiments we used a lab scale cassettes which has a membrane area of 50 square centimeter. It has the regenerated cellulose ultra cell 300 kDa membrane. We do have a section. In the following sections we'll cover how we can scale up from this 50 square centimeter up to a up to 1.5 square meter filter area. So. This graph kind of shows how the TMP and flux profile evolved during concentration and difiltration. So the X axis is showing the processing step. As I mentioned before, the 10X concentration is achieved through two concentration. First it concentrates to 4X and then we do A5DB difiltration. Then we perform the 2nd concentration which takes us to 10X. The primary Y axis shows the permit flux. Again, that's more for the TMP control, and the secondary Y axis shows the TMP and that's for the permit control. For the SO first let's look at the TMP control which is shown. The curve which we are looking at is the blue one where the TMP is fixed at 8 psi. Once you set it then you leave the process to naturally to decline. So as you can see the flux declines as the feed concentrates and that's because. As as it concentrates, the wall concentration increases and the membrane starts to gradually fall and it is indicated by the decline in flux for the TNP control. During difiltration the flux remains fairly stable and then during all during your second concentration step it again further the flux again starts to decline. In contrast, for the permit control, you do have have to set your flux level for each step. For your first concentration step you set it at 55 lmh and as you can see, as the membrane starts to gradually fall, the TMP starts to rise. At your dye filtration you cut the flux by 50%, which brings the TMP down and remains fairly stable during dye filtration. During your UF 2, you go back to your. Initial concentration flux that led to a rapid rise in TMP where we had to like cut cut the flux by 50% for TMP to be lower and so that it would complete and stable condition again when you compare the TMP control and the permit control. TMP control had this lower risk of exponential rise in pressures. Next we'll look at a case study using the A AV Street AV stream. Specifically this is the a A V2 server type where we compare the TMP and the permit control. This was run at 80 liter per meter squared loading and can maintaining A5 LMM cross flow flux as you can see from the graph. The average flux and processing time. The TNP control had the advantage of flux and processing time. That is, because you're in your TNP control, the flux is always covering close to the limiting or the plateau region of the flux. Whereas in the permit control you are setting the flux at a conservative 50 to 75% so that you don't have that rapid rise in in your TNP. So, so, so again, there's a there's a clearly a flux advantage when you use your TMP control over the permit control when you compare the transmembrane pressure. And since you're restricting the pressure since since you're restricting the permeate side, your your TMP is lower for the permeate control when compared to your TMP control. But again, both. Processes have there within the limits that we had specified initially of 15 psi. Next let's. Next we're looking at the a AV yields the recovery from both TNP control and permit control where very similar close to 90% and then the impurity removal of. We track 3 impurities HCPDNA and Benzonase and in all three cases you can see that the permit control had a. Slight advantage over TMP control. Again, for the permit control you do have the control of the flux. You're able to control the polarization layer which gives you better sieving of impurities when compared to the TMP control. So to overall, to summarize, TMP control is a simpler system, it just requires one feet pump. It also would. Run faster than the permit control and in terms of recovery they are very similar to the Permit control. For permit control you can run the process at lower lower Tmps and also it has the potential to clear higher impurities in your TFF one step the next. I would like to move on to the scaling performance of single use. TFF filters. So for as we saw before, for the lab scale we use the 50 square centimeter cassette and for manufacturing scale we go. We start using, we start preferring the Pelican capsule that are designed for closed processing. They they come and plug and play format so they do not require a holder unlike the like. Like the cassette where you have to torque and you you you need a holder to to have the filter intact. These these capsules are come gamma sterilized so they're ready to use in minutes, and they can be scaled. They're linearly scalable, down from 50 square centimeter all the way up to 18 square meters. This is a TFFTMP control experiment where we perform the flux versus TMP excursion of five different device sizes. The 50 square centimeter cassette .1 square meter capsule, .5 square meter capsule, 1 meter square capsule which is essentially A manifold of 2.5 square meter capsule, and the single largest capsule which is the 1.5 square meter capsule as you can see for both 100 and 300 kDa. Membranes The flux performance is very consistent across different scales, leading to predictable performance. This is a case study, which again we did with the a, A V2 serotype where we're trying to demonstrate scaling between A50 square centimeter cassette and the point squares .1 square meter capsule, the feed of the. The feed of the cassette was generated in a shake flask and feed of the capsule was generated in a bioreactor that resulted in 4.6 Volt higher titer for. For this study we used a permeate control process at 16 liters per square meter, loading again the same processing step of 10X concentration and five DV dia filtration maintaining up five liters per meter square per minute cross flow flux. Since they were run at permit control mode, they both the cassette and the capsule had similar average flux and processing time. The TMP was slightly higher for the capsule when compared to the cassette and that's due to the higher titer of a AV feed that was generated in the bio reactor when you compare the flux. Or sorry, when you compare the eels, you can see that both the XL50 and the capsule had high eels. The capsule had eels close to 99%. And then when you compare all the different impurity removal performance, you can see both the XL50 and the capsule had similar performance. So overall Telecon XL50 cassette predicts the performance for scale up with the telecon capsule. With that, I would like to conclude my section and hand it over to Rosario, who would be covering the TFF module comparison section. Thank you. Thanks on very much. As on Instead, we'll be comparing spirals and cassettes and hollow fibers from module design to performance during. TFF and also range of module performance. So the hollow fiber, fairly simple module, fairly simple filter. It's a tube, you have the feed going through the tube and the permeate going through the walls of the tube. Whereas the flat sheet cassette and spiral wound capsule, they're a little more complicated design. There are some similarities and differences between the two. They both have a feed screen, so the speed screen here serves a couple different purposes. It separates the membranes and keeps them from compressing onto each other and it as well promotes uplift of material away from the membrane surface which helps increase your permeate flux. The main difference between the flat sheet cassette and spiral wound capsule are in the name. So the the cassette is the flat sheet cassette is indeed flat and the spiral wound capsule is wound around a permeate collection pipe. The core here a slight difference in the flow pass for these two. The capsule has this counter current flow where the feed travels through the device, the permeate goes in and the flat sheet cassette can be set up in multiple configurations. So the shear stress in a device is one of the main stresses of products. So given that the hollow fiber is a fairly simple geometry device, it's also a fairly simple equation for understanding the shear stress. It depends on the average velocity and a single fiber and your fibers in a radius, so this average velocity inside a single fiber can can change between. Determine, based on the length of your device, the area of your device. There's a few different factors that can tweak that velocity. For the flat sheet cassette and spiral wound capsule, we've designed them in a way where they have very, very similar performance and so the sheer for these devices, the cassette and the capsule are given by the type of screen they're using. So in this case we're looking at devices with a C screen. And this is determined by the shear stress is determined by the height, the feed flow and the average feed viscosity. The computational simulations were performed to understand the the shear stress between different device screens and it's and it scales between the flat sheet cassette and the spiral wound capsule for the same cross flow. You should expect the same shear on your product. Now we'll look at an assessment for TFF One with our AAV 2 model stream. Here we're using again the Pelocon XL50 cassette, the scale down tool, the Pelocon 0.1 meter square capsule and polyfibers. So we used permeate control for this comparison. We wanted to test the wide range of feed cross flows device configurations and have access lower TMP's and to reach that critical flux of the device. Here the loading for each of the module tested is 35 liters per meter squared and our Model T FF1A a V2 feed is non transduced Heck 293 which has been lysed Benson ace treated and clarified. And we used Phi X174AS our tracer particle. So here we evaluated the effect of doing a critical flux excursion going in two different directions. So we have an increasing this excursion, this triangle. So we started at 3 lmm and went up to seven LM. So after we reached our critical flux at three we. We would change our cross flow flux to five element, find our critical flux and then go to seven element. We also looked at a decreasing excursion. This decreasing excursion started at seven element. We found our critical flux. Then we went to a cross flow flux of five element and then down to three element. What we found here is that the subsequent critical fluxes that you test after your first do you have an effect on. Your critical flux that you measure. So your first critical flux will likely be the best that your device can achieve. The subsequent critical flux as you measure, may have a degradation from the original critical flux, so the order in which you perform them does matter. However, we found very similar performance at the second critical flux that we tested for both devices, so taking that knowledge, we. We understood now that we need to start our critical flux excursions at the cross flow flux that we want to perform our process simulation. And so here we're looking at a Pelican capsule and a Pelican XL50 cassette. We both started at 5 LMM cross flow flux. We had the same critical flux between the two formats and at 7 LMM we had less than 10% difference between the two and again the same. Critical flux at two element, we can add 300 kilodalton onto this chart. So this is 100 kilodalton devices. When we add 300 kilodalton onto this chart, we see that we have a slightly higher critical flux at five element, similar range at Seven element, so little higher than that for the Capsule for 100 kilodalton versus 300 kilodalton. And again here for the capsule at 300 kilodalton, you have a slightly higher critical flux than the 100 kilodalton. And with the more open membrane you are more susceptible to fouling which is causing some of this decrease in critical flux from expected. Here we can put on the chart all of the devices we tested, all of the Pelican devices we tested here and all the hollow fibers we tested here. Hollow fiber A in this plot is purple, Hollow fiber B is green. The different symbol shapes represent different lengths of the hollow fibers. As I stated earlier, the different lengths of your hollow fibers in different areas will determine the average velocity through your device, which changes the shear stress in your device. We found here is that the highest cross flows we tested for our devices. And the and the hollow fibers, we still had A2 to 3X permeate flux benefit. So our highest permeate flux is achievable for the Pelican devices still about two to three times higher than the Pelican filters. So for the next section that we'll get to in a minute, we tested these specific we use these specific cross flows for each device for the Pelican capsule and Pelican XL50. Around 130 A, 130 LMH and 110 LMH for hollow fiber A and hollow fiber B5 LMM and 15 LM. So we spoke earlier about shear stress in the device. So we can actually change this plot. We'll keep critical flux on the Y axis and we'll change cross flow flux on the X axis to shear rate that the product is experiencing in the device. When we make this change so polyfiber A and hollow fiber B fall on the same shear rate around 6100 inverse seconds and the pelocon devices at five element are approximately 5100 inverse seconds or even at lower shear rate, the pelocon devices due to the screen that promotes uplift of material away from the membrane surface are able to achieve. A higher permeate flux in the hollow fibers that we had tested in this study. Here we can look at the permeate flux versus TMP. So this is again going to what on it had shown. Instead of looking at the the permeate flux as a function of time, we're going to plot it similar to what A11 pump TMP control experiment. So we have trans membrane pressure on the X axis, permeate flux on the Y axis. We see here with the Pelican devices that the TNP versus flux scaled very well between each other. And what we find with the hollow fibers is that whenever you start to approach your critical flux, the TNP rapidly rises. So these readings for the hollow fibers are about 1:00 to 2:00 minutes apart. And you have to really pay attention once you start to approach these instabilities and to put that plot into more broad terms, we can look at just the critical flux of each device compared to the capsule. Once again, here we have permeate flux, normalized the capsule. It just demonstrates again the higher fluxes that are achievable with the Pelocon devices that are still gentle to the product that you're focused on. Now we can look at the 300K dolton case study that we did. So the process simulation where we did T FF1, we did ultra filtration for our first concentration one to four X $5 die volumes die filtration and then our second concentration from 4:00 to 10:00 X. We did this in permeate control, so we used the pump on the permeate. And for the, as we outlined earlier, the feed cross flow fluxes telecon XL50 and capsule we use 5 lmm and the two hollow fibers we used 5 lmm and 15 lmm, about 6400 inverse seconds here we're going to process the, we're going to track the process stability during each step we'll walk through the caps or the Pelican XL 51st. Then we'll look at the capsule and see how the TMP during the process scaled with the capsule and then we'll add in the hollow fibers. We have a linear increase in TMP during our first concentration with a fairly high flux at 55 LMH. During diet filtration, we decreased our flux from our original 50% of the critical flux to 25% of the critical flux. We had very stable TMP during diet filtration and as on it has showed earlier. When we went back to our second concentration, we went back to 50% and found this instability and so we used that to infer our future experiments. So we decided to do our future experiments at 25% of the critical flux at during our U F2 step. So using that information we tested again the capsule. So a little bit higher TMP during each of the steps, but it's also given the higher flux that we use during the process. Important to note that scaling tool, the XL50, was able to allow a stable second concentration step given that we were informed of the lower flux required. We add hollow fibers onto this plot. A fairly stable DMP through each of the steps during our first concentration diet filtration and our second concentration. Something to note here, the green, the green lines of for hollow fiber B, we had to in the process short given that we reached the minimum system volume. This occurred due to the fact that we. Boost the performance of the hollow fiber beam. We had to increase the feed crossflow which required larger tubing for the system. So this larger tubing increased the system hold the volume and we were unable to reach our targets. So next we use the fluxes during the process to inform a model to understand how. These different systems would behave given couple different scenarios. So for this system sizing analysis we look at a small batch 10 liters and a large batch 200 liters at the same loading. Main take away for here is that time normalized the capsule you're going to have about A2 to 3X time benefit. Pelocon capsule and hollow fiber A. Given the V cross flows used, you have the same maximum volumetric concentration factor. But the capsule is able to complete this step in a faster times time span and the same relationship showed up for hollow fiber B that we had inside the lab. Is that the maximum volume electric concentration factor? Was affected by the system sizing. So by using this higher feed cross flow it did limit the system on both the 10 liter and 200 liter scale and it showed for 200 liters that you would not make it to your target to next concentration. The next sizing scenario we look at is. Constraining our process window to two hours. So we have a 2 hour process window for a small batch 10 liters and a large batch 200 liters. And a similar relationship crops up when you constrain time. So prior we in the previous scenario, we constrain the area between each device used. Now we're constraining time and a similar relationship shows up that when we normalize time required to the capsule. We find that it takes about two to three X less time to complete the process simulation due to the higher permeate fluxes that are achieved with the screen device. The larger areas required for the hollow fibers may have some significant effects when it comes to. To hold the volume of the filters that you're using, the increased buffer and flushing requirements for conditioning a larger amount of area which can lead to an increase in cost for a production run. Now we have our final poll question for the audience. Would you be interested in using a Pelican capsule, A for yes, B for no, C for maybe? All right. Thank you everyone for your responses. We really appreciate the feedback. And so now we'll summarize what Anand and I have spoken about today. For the Pelican capsule and the hollow fibers we tested, we demonstrated that the Pelican XL50 cassette serves as a very effective scaling tool for the capsule. Also provides a solid starting point for process development with viral vectors. It's also shown that TMP control simpler and faster to implement, while permeate control was able to keep pressures lower in general and resulted and generally higher in purity clearance. There's a trade off with that. The Pelican filters we showed today demonstrated higher permeate flux than the traditional hollow fibers we tested. Which resulted in faster process times. Also, Pelican filters benefit from low shear and low cross flow flux. This is ideal for sensitive biologics and have economic impacts down the line when it comes to production. Thank you. Thank you Anan and Rosario for this great presentation. Now it's time to answer a few questions that have come in from the audience. But before we do, I would like to remind you that it's not too late to send us your questions now using the Q&A widget. This also applies to our on demand viewers. We will try to get through all of the questions, but if we run out of time, we will respond to you individually. As a reminder, this webinar will be available on our website shortly. All participants will receive an e-mail notification when it is available for viewing. Now back to Annan and Rosario, who will start answering questions that have come in. I just wanted to say thank you to everyone involved with this. It takes a village. There was a lot of people that helped make this possible. I just wanted to give a big shout out to everyone that helped us along our journey to acquire all this data and knowledge. So with that, feel free to put any questions into the chat. I'll take the first question here. Can you use flat sheet screen devices for sheer sensitive applications. So in in our application note with the that we we've published with these devices we looked at lengthy virus as well and we found that you could process lengthy virus with with our devices. The screens promote uplift of material away from the membrane surface and are fairly gentle on your streams. To be even safer, you could use a lower feed cross flow. So instead of five element cross flow flux, you could decrease it to about four element cross flow flux. But we found an insignificant effect of our devices on recovery. All right, we have the next question. What are the main advantages of permeate control operations? What are the? Disadvantages of permeate control So advantages I would say you have more control on the process. Again you can control the membrane polarization and falling by setting the flux you want. This has shown to give give you improved sieving which in our applications would provide better impurity removal like benzonase and whole cell proteins and DNA. The other advantage is the lower TMP you can operate the process at. So the TMP is basically the difference between the feed side and the permeate side. And when you restrict the permeate side you achieve lower TMP throughout the process. So that's the advantage. Disadvantage I would say it's it's a more complicated system. You need an additional pump valve control system to run this run run the puppet control. The other disadvantage is the pressure rise as I showed any variation in upstream, like the example where the tighter values were higher is our rise in TMP and in some cases you have to interfere to reduce the flux, but in case of TMP control, you set it once and you don't have to worry about the rise in pressure. All right, the other quest, the next question is does impurity removal scale between capsules and cassettes? But yes, the impurity clearance will scale. The example that we showed today had the same ultrasound 300 kDa membrane on both device formats and it had similar processing steps of 10X concentration and 5DB DI filtration. And they had similar impurity clearance. So the answer is yes, for the same membrane you can use the cassette or the spiral. You'll get the same impurity removal performance. So we have another question, Could you explain how the graph critical flux versus cross flow flux was generated? So I'll explain this in 2 steps. So in this plot here, we have our permit flux versus TMP. So each of these markers at a specific flux is in five minute increments. So we'll hold our our flux for 15 minutes total. We record our initial flux at a specific TMP and then we observe the TMP. Either stay stable or increase. So in these cases, so say here we start to observe holding the flux constant, our TMP starts to increase and we define the critical flux as a rise and average rise of 1.5 psi over 10 minutes. So that criteria was met here at this flux. And so we quantify our critical flux as the average of these two fluxes. So if we look at, if we look at the hollow fibers, that would be the average of this flux flux, that would be your critical flux. That's how all these critical fluxes were determined. Slowly increasing your permeate flux and observing your TMP stability at specific fluxes until you reach an instability. And you might be able to answer, yeah, there's the next question is have you ever observed product quality impacts are running in TMP versus permeate control modes. This, this is a good question is for TFF one before the chromatography went, the stream has a lot of impurities, whole cell proteins and DNA. We haven't seen any product degradation when you run in TMP control and permit control mode. But I believe there are studies in the literature that show that higher flux in for the TFF 2 step that is post chromatography before the for the formulation. You do tend to see higher product losses when you run it at higher flux. So that's something which we have not internally tested and we plan to do that in the future. Again for the, for the, for the, for the T FF1 part before the chromatography we we do not expect any product related degradation when you use TMP or the permit control. Any other questions? Anand and Rosario? I believe that's all the time we have today. I would like to thank everyone for their questions. And if we did not get to your question, please feel free to e-mail one of our presenters directly. To register for future webinars or to access our archived webinar library, please visit our website. I'd like to thank Anand and Rosario for today's presentation and thank you to the audience for joining us. We wish you a great day. Thank you everyone. _1733894454579