Hi, everyone, and welcome. My name is Mohammed Aoudi and I will serve you as your moderator today. Thank you for joining us for today's webinar, Single Use Scale Down Predictive Solution for Intensified perfusion development. As your moderator, it is my role to ensure that you make the most of your time with us. I'm here today with Kevin Lee and Long Fun. Kevin Lee recently recently joined the company with the acquisition of RB Biosystem. Kevin was a cofounder at Arabi and developed the core microfilm technology platform as a part of his doctoral research at MIT, including design of microfilitic continuous culture system and plastics consumables manufacturing. He is also an expert in embedded systems, cell culture and bioengineering. And holds a BSE from the University of California, Los Angeles and a PhD for the Massachusetts Institute of Technology. Kevin He's an inventor of six patent and led programs with industry and academia across multiple cell type including E Coli, Pica Show and Cortese. Dr. Kwong Long Phan, Senior Scientist in the Innovation Cell Culture Media Developing Group, focus on delivering the fusion products. And process development is currently leading a high throughput microfluidic platform technology skeletal model for perfusion processes that significantly enhance the capacity of perfusion medium development. Before joining the company, Doctor Farm earned his PhD degree in chemical engineering from New Jersey Institute of Technology and received a post doctoral training in biomedical engineering at Tufts University. So before I turn things over for our presenters, I would like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets you can use. There you can also find our reaction button indicated the thumbs up image that allows you to give you immediate feedback on the presentation topics or anything that stand 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 WeChat. We'll 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. We will also have the important to participate. A couple of quick all question throughout the session. I encourage you to take part of the Indie survey if you are watching this webinar on demand. You can still submit all responses and questions and questions via the Q&A WeChat. The webinar is being streamed through your computer so there is no dial in number for the best audio quality. Please make sure your computer, speaker or headset are turned on and the viewer is up so you can hear the presenters. Nondemand 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 was a pleasure and I will turn things over to Kevin. It will start presentation. Thank you Mohammed for the kind introduction. I'm excited to introduce to you a single use scale down predictive solution for intensified perfusion. Development I will start by introducing the Breeze platform and then give some details about the technology and how it implements all the functionality of a perfusion bioreactor system. So this is the Mobius Breeze Microbi Reactor perfusion platform. The system contains 4 independently controlled bioreactors that we call pods. Each pod operates 1 microbi reactor at A2 milliliter working volume. Underneath the four pods is a base station. And a CO2 sensor box which handle facilities, gas distribution, measurements and communication. A laptop and with controller software is also provided to monitor, control and record process data. The single use consumable shown in the center operates at A2 milliliter working volume and contains both the microfluidic cassette and the fluid bottle assembly. The whole assembly is gamma radiated and the single use assembly contains the entire wetted fluid path including the perfusion filter, pumps, valves, sensors and mixers. So what can you achieve with the Mobius Breeze Microbiome reactor? The Breeze is an excellent tool for building knowledge of your profusion process. Small working volume enables profusion screening during cell line development, media screening and optimization, and early process development without need for significant resources. This allows you to get more data earlier in the development cycle and hopefully speed up development times. Next, Mohammed has a pull question for the audience. So the first question is which of these best describes your experimental works for so select the best that apply to the situation a the fusions in line development B the Fusion Media screening and optimization, C Early process development for perfusion processes D not currently working on perfusion. OK. Thank you for answers. I will give back the presentation to Kevin Mohammed. Can you try turning your camera off? It seems that the audience is seeing your camera instead of the slides. Interesting. I'm seeing this slide, Kevin, so you are. Yeah, keep going ahead. Thank you. Sorry for this delay. OK. So the workhorse of the system is the single use consumable assembly. This assembly comes gamma radiated and contains both the microfluidic cassette as well as all the tubing and bottles required to operate it. This ensures that the entire wetted fluid path of the system is disposable. Quarter inch ODC flex lines are available for all inputs, including the four input feed bottles and the inoculation line. There is a sampling line T to the inoculation line so that there's only one cell input output port to the reactor. The source bottles are also pressurized with .2 Micron filtered air, minimizing contamination risks. The input bottles are consistently pressurized with gas containing CO2 to control the pH of the media bottle. In addition to the input 2 welds. There is also a needleless port for a septic fluid removal, operated similarly to the process of sampling a larger bioreactor, but aided by consistent bottle pressure to help drive out the liquid and maintain sterility. The perfusion bottle has a small reservoir which collects the harvest into 1.7 ML tube before overflowing into the main bottle. This tube can be emptied prior to harvest collection to ensure that the harvest concentration is representative of the reactor when sampling, even at volumes as small as 200 microliters. The waste bottle collects priming fluid as well as cell bleeds, and also has a vacuum connection to enable automatic line priming during inoculation. All of these bottles have a 60ML working volume, enabling 30 days of operation before needing to refill any fluids when running a one BBD process. In the next section we'll introduce the microfluidic cassette architecture and its features. The top view of the cassette is shown on the left. The input, the fluid inputs and output bottles interface to the cassette from the left side. Each of the four input bottles fills one of the pink Oval fluid reservoirs on the device. One of these reservoirs is reserved for DI water and the other three can be independently programmable fluid inputs. The four independent input feeds are connected to a shared peristaltic pump, so flow rates are consistent between all lines. The integrated peristaltic pump connects to the growth chamber, which is shown in blue. Inside. The growth chamber contains an embedded 1.2 Micron PDS filter. And a second peristaltic pump downstream of it to pull perfusion harvest that is integrated into the device as well. There are a variety of optical sensors similar to what you would find in an AMBER 15 consisting of DO&PH sensors. Additionally there embedded sensors for optical density would provide online information related to cell density in real time. Since the fluid control and sensing is integrated within the same device, operation is simple. There are no separate auxiliary configurations or equipment needed. To enable high density perfusion culture, if we look at a cross section of the device, there's a thin 100 Micron silicon membrane sandwiched between 2 rigid plastic layers. The bottom layer has channels cut out for all the fluids, whereas the top layer has air pockets placed in specific locations to create active structures. By introducing pressure vacuum into an air pocket, you can effectively deflect the silicon membrane to laminate against either the upper or lower layer, effectively forming a pinch valve. If we follow the structure from left to right, we can trace the path of how media is injected into the reactor. The bottle input fills the reservoir with media going through the input valve and then the channel select and chamber valves are opened and the input pump is used to drive liquid into the growth chamber. Larger deflecting membranes are connected together to form the growth chamber. Here, the actuating gas above the chambers is used to both provide control of the concentration of oxygen and CO2 in the reactor, as well as provide the driving force to move fluid between the chambers and perform mixing. In the next slide, we'll show a video of how this system mixes. Here we have channels filled with green dye. The cassette injects approximately 500 microliters of green dye into the chamber. Then it turns on the mixer to mix the dye through the chamber. Processes shown in real time, and you can see it takes only about four seconds to fully mix the liquids together if we wait. So it should happen a second time so you can watch it do it again. OK, so let's look at how the mixing actually works as a gas pressure fills the one chamber on the left. The membrane stretches downwards, which results in the pushing the fluid into the neighboring chamber on the right, this chamber's head space volume shrinks and pushes the waste gas out. As this process repeats back and forth, the pot is actively mixing oxygen and carbon dioxide gas into the pressurized gas that sources the mixer, which is in response to the pH, CO2, and DO sensors on the system. Since the silicon membrane is only 100 microns thick, diffusion is a fast enough process that high KL A's upwards of 30 to 40 inverse hours. And gas transfer can be achieved without the need of any bubbles. If we look at the diagram on the upper right, we intentionally operate the mixer deflecting in the high frequency state where the membranes move quickly and do not have time to fully inflate and contact the bottom of the growth chamber per stroke. This contacting action would be similar to cells moving through a two through tubing of a perissaltic pump and would result in very high shear, which is a situation we would like to avoid. In addition to frequency, we can also control the KLA with an intermittent delay, as shown in the block diagram below. This simply means turning the mixer on and off periodically to reduce the amount of mixing and gas delivered. So how does integration of all these components into a device help with automation? Integration allows us to run on a single control platform where our software manages all aspects of the Buy React control without any requirement for third party software or hardware. Our software has real time monitoring of all online data via plotting interface as well as integration of system alarms directly into the software. We also implement A variety of Labor saving automation tools and tasks to help streamline the process such as Handsfree, DO&PH calibrations, active volume management. Close of control of all the online sensors and software programmable fluid gas delivery and sample removals. The microfluidics also gives us precise control so you can trust the process. Each reactor comes shipped with precalibrated pumps that are barcoded and complete. Isolation is possible between all the different reactors in the four pot system, so you can run for independent cultures at the same time. They don't even need to be the same cells. So volume management is possible in the two milliliter working volume because of the microfluidic architecture. As long as the silicone membrane has only two states, either fully laminating the upper or lower rigid walls of the valves, then the volume of any valve is defined only by its respective plastic cavity volume. If we look at how the parasol pump works on the left we have 3 valves that operate to move a discrete plug of liquid from the left to the right. Liquid is sucked into the center valve which has a volume V. Which defines the injection volume. This plug of fluid is isolated when the incoming valve is closed but the outgoing valve is open. Then the fluid plug is ejected to the right. Each time the cycle occurs, we inject precisely the volume V so all fluid flow can be discretized without the need for drift correction or recalibration. Growth chamber inoculation, volume management and sampling also occur in a similar way, this time using the growth chamber as the valves in this cartoon example with only two chambers. The reactor operates with one chamber full and one chamber empty. During inoculation, we start with both chambers empty. Then we pull vacuum to draw in the inoculum until one chamber is full. This process automatically stops on its own. Since the membrane can no longer inflate once it has hit the top surface during sampling, we can reverse the process. First, we predilute the growth chamber to increase the working volume above the nominal volume, in this example by about 100 microliters. When we when we reconfigure the reactor in the inoculation condition, you will notice we now have an extra 100 microliters trapped underneath the section that should be empty when we open the output port. That allows the membrane to inflate all the way to the bottom, ejecting any excess liquid that was in the chamber. This corrective action can also be performed to the perfusion port periodically to guarantee that the reactor volume does not drift over time, and by using this process we can eject. Sample volumes as low as 50 microliters very accurately. Another labor saving automation task we talked about is sensor calibration. Instead of precalibrating sensors prior to arradiation, which can succumb to poster radiation drift, we run an automated multipoint calibration procedure poster radiation in because that's we're going to use instead of injecting sterile buffers directly into the chamber. We can take advantage of the media's bicarbonate buffering capability to change the pH only through gassing. How does this process work? First we need to build a pH versus CO2 reference table for a particular media using a supplied pH electrode and sparking setup on our system as shown on the left. Using this we can gas the CO2 into the media and measure with the pH probe to build a table that's shown in the middle of CO2 and pH. This look up table can be used for further calibration. Once we have the table, we can run the same media inside the cassette, then measure the raw sensor pH data, measuring the predefined mixtures of CO2 with our CO2 gas sensor. Once we have the sensor and CO2 data, we can convert the CO2 directly to pH using the lookup table we previously generated. This gives us A4 four point calibration which fully characterizes the pH sensor response for any cassette and doesn't require the user to do any offline sampling for the process. We talked about how high gas diffusivity of the silicone membrane helps us achieve very high KLA. Unfortunately, the gas transfer rate of water vapor in a small scale culture system like this is also very high in in our case, we can lose as much as 37% of water per day through evaporation. Since our geometry and air liquid interface is fixed, we've characterized the expected evaporation rates under varying temperature and mixing conditions. This allows us to automatically correct this in software with the I water injections so the user does not need to worry about evaporation induced concentration drifts which can Co found small scale experiment data in our system or other small systems such as Wellflins. Another key advantage of our geometry and mixing strategy is our ability to integrate turbidity sensors directly into the cassette. This is mainly possible because the geometry is fixed and there are no bubbles that are present for aeration. In the cell density configuration, we fill the sensing chamber completely and measure the turbidity through the cells. Next we can also reconfigure to push all the cells out of the sensing chamber to create essentially a Cuban blank. These two measurements improve the stability of the sensor to any common mode drift like surface felling or radiation induced color change or anything else. Our sensor comes with two path links as shown. On the right is a calibration curve for Cho cells, demonstrating that even the longer path link OD sensor works out to 200,000,000 cells per milliliter. Keep in mind that this is only a turbidity measurement which is related to particle scattering, so it is more analogous to total cell volume than it is to cell density. But for many perfusion processes, cell viability and diameter stay similar and high for the whole process, which allows us to use this as an analog for a predictor for viable cell density. Since the optical density sensor is integrated directly into the reactor, we can measure cell density very quickly, enabling simple but precise OD control for a fixed perfusion rate. When the density is large in this set point, we simply divert the perfusion outflow to the waste bottle bleeding cells, and when the density is less than the set point, we can resume perfusion as normal. Below our graphs demonstrating the consistency of this type of control. On the left is the online data showing the optimal density for four separate pods in a steady state profusion process. And on the right shows the offline cell counts over the same 24 day period. Great. So next I think Mohammed has another poll question for the audience. Thank you, Kevin. So the next question is what is your biggest need in upstream processes process development? A ability to run parallel experiment for more data high throughput, B integrating process analytical technologies, C easy to use system with less human interaction labor, B adequate model to predict performance of large scale bioreactors. So now I will let the audience submit their answers during a few seconds. Thank you for your answers. Now we'll give the floor too long for the rest of the presentation. Thank you, Mohammed. Hello everybody. So today I would like to give you an idea about how we leverage the Breeze technology to improve our. For Fusion Media development workflow then I'll be showing you that the Breeze is highly predictable to other bio reactor skills and then I'll be share with you some of the application data that we generated using the Breeze during our couple of for Fusion Media developments. So first I would like to give you an overview about different Bio Reactor models that we have been using. In our cell culture media development for provision processes, so there is a wide variety of different scales ranging from 1,000,000 liter to three liter. At a lower end, you can see that we have a 96 liquid plate and TTP speed tubes. So these two models are very popular. They have high throughput capability. So very suitable for a screening phase which happened at the early stage of the development process. However, they don't have a control environment. The larger scale bio reactor such as the Mobius, the single use and the applicant bio reactors have a tight control over the culture environment, but they don't have a high throughput capability so they are suitable for. Confirmation runs which happen in a later stage of the development process. We also use Amber 15, Mobius Breeze, and customized single U profusion bioreactors. The Amber 15. As you may know, they can only perform simulated profusion because they don't have a true profusion capability. The Breeze, on the other hand, has a true profusion capability. And had a high level automation, so it is very suitable for our perfusion purposes and potentially can help us significantly improve our perfusion media development workflow. So in the diagram I'm showing you here is the current workflow we use for the cell culture and media development dedicate for perfusions. So the process up from the left to the right. We're starting out with the screening and finally and with the final prototypes. So this workflow are predominantly used in the fat bash product developments in the past which involve a different vibrator scales including the 96 bit web plate, the TPP spin tube DM-15 and also the large scale bio. We have to spoken informations and our pinpoint here is that. Because the small and middle scales bio reactors don't have the true perfusion capability. So the data that we generate not very well correlate to the larger scale bio reactor. So that's requires a large amount, a large number of confirmation runs using the large scale bio reactor which have a very low throughput and as a whole the developmental process is lengthy. Convoluted and can take over a year to complete. So we want to propose a new workflow in which we want to use the Breeze to replace some of the small scale and middle scales because the Breeze had a high total capability. So it can be used in a very early phase of the development process and it's also can generate the data comparable to large scale bio reactors. So we can. Reduce the number of confirmation runs and therefore as a whole we can streamline and significantly reduce the development time. So here are the few number I want to show you about the advantages of using the Breeze. Compared to the band scale bio reactors, the Breeze has much higher throughput and why? We can help you save time on the experiment setup and clean up it also significantly. Reduce the amount of media usage at a cost related to that up to 1000 time reduction in the media and region Asian cost. So next I would like to show you that the Breeze actually fits our perfusion purposes. So here I want to give you an some fundamentals about like different perfusion modes that we can perform using the breeze. Including a dynamic and steady state perfusion. So what are dynamic and steady state perfusions? Both of these process involve the cell retention device to keep the cells inside a bioreta at own time. So while in the dynamic perfusion, VCD is allowed to grow as high as possible without any cell bleeding, in the steady state mode the VCD is set at a certain value. And that value is maintained constant over time by bleeding the cells out to the the certain vessel or container. So the pro and cons of each process here can be the cons and pro for the other process. For example, in the case of a dynamic profusion, it has a short counter terms less complicated in setting up and can achieve a high VC as well as the high titer. But the viability of the dynamic pollution drop early and we have more ways accumulated and also the quality can be affected by the high level of waste accumulation as well. So in the case of steady state you have a more stable process and culture. Longevity can be maintained better and lower waste accumulations, but it's actually require a longer longer culture terms with a complicated setup. And you also have some kind of product loss through bleeding. So using the priest we could generate the data for both the dynamic and the static state profusions. So for the dynamic profusion culture, the two graphs and the top two graphs is showing the data that we generated in a dynamic profusion culture of a certain 2 clones at 1.5 vessel volume exchange per day we could reach a 150 million cells. Density with the viability was maintained at a reasonable level and the volumetric productivity in the QP was increased over time in a dynamic perfusions which in the QP could reach around 20 people gram per separate and the volumetric productivity was around 2 gram per little per day level in the case of steady state perfusions. The data was shown in the bottom 2 graphs. On the left hand side, you see that the VCD was maintained at a stable level of 50 million cells per mu. And here we test 1.5 and one VVD, two different profusion rate and you could see that at two different profusion rate, the VCD was maintained at the constant level and the viability was very well maintained both 90 to 95%. Both the volumetric productivity and the specific productivity was maintained at a very stable level. In the steady state profusion culture. So next I would like to show you that the breeze is highly predictable to other scales. So there are certain number of metrics we want to use. In this comparison we focus on the self growth which is the Vcity and viability and also the cell productivity which are tighter. Volumetric productivity and sales specific productivity. So the first piece of comparisons, the Breeze was evaluated together with the three liter glass Bio reactor which is a standard bio reactors. So here we compare the Breeze to that that's still a glass Bio Reactor at different perfusion rate from 0.5 Vd to two VDD. The. Self growth and the specific productivity are put into the comparisons. So on the left hand side the graph was showing the self growth and on the right hand side is a specific productivity. So looking into the self growth you could see that there is a great correlation between the breeze versus the glass bioreactor in own profusion rate. The only exception here is the viability in the breeze is higher was maintained higher compared to the larger scale bioreactors. Properly owing to the lower shear stress in the breeze environment and also the breeze is operated in a bubble free manner, so it can be free from the bubble induced shear stress and therefore can maintain a higher viability compared to to the glass bioreactors. And in the case of the specific productivity we do, we don't observe any significant difference between the two. Bio Reactor scales suggesting that the Breeze is highly predictable to the band scale bio reactors we also have another comparisons. So here the Breeze was compared to the TPP speed tube and the band scale glass bio reactors. And here the metrics we use is the different process parameters compared to the band scale glass bio reactors. The Breeze match on of the. Process parameters are required for a bio with the typical bio with the runs, but in the case of the TPP speed tubes, since it doesn't have the control environments and can only run more for fusion. So we might expect that there's certain difference between the TPP speed tube versus the other two bio with the scales. And one thing I wanted to mention here is using the Breeze you can inoculate at very high salinity up to 20 million cells. Per milliliter, which is around 10 times higher than the event scale plus bio reactors. So with that highest cell interpolation density you could reach the basically set point earlier than the band scale. Bio reactor. I put in here some of the comparison there regarding the tighter and specific productivity for certain chosen clones. So compare to the glass bioreactors, the Breeze is better comparison than the the TPP spin tubes In the in case of a tighter you can see that the tighter dip between the TPP and the glass differs by 20%, but it's like around 10% different between the Breeze and the glass. There was no difference in the specific productivity between owner. Different scales here, suggesting that the Breeze can be very highly comparable to both scaled out and larger scale bio reactors. In the case of the specific productivity, we also compare the Breeze to glass bio reactors of different size, and here we compare across different cell lines including the. Chosen and a choke one in our two catalog Perfusion Media including the 4X expansion media and the XM Advanced HD Perfusion Media. So if you can see in here in all the chosen clones, the data is showing that the Breeze is highly comparable between comparable towards the glass bioreactors. There is a slight difference in the case of the choke one SV, sorry the choke one. But what we see in here, the difference here is minor according to our experience. So next I would like to share with you some of the application data that we generated using the Breeze when we develop catalog for Fusion Media. So the parameters that we want to use the breeze to identify is the cell specific perfusion rate. So what is cell specific perfusion rate? Is it abbreviated as CSPR? So the CSPR is defined as the amount of media perfused through a single cells per day. And it is calculated by dividing the provision rate by the viable cell density. And so the two illustrations show in this slide, on the left hand side is dynamic profusion and on the right hand side is static profusions. So the CSPR profile of dynamic profusion differs from the steady state profusions. And in both the graphs here showing that the CSPR is. Profile is shown in the black dash line. So in the case of dynamic perfusions, because the VCD is allowed to grow to the maximum level, so at the constant perfusion rate you can expect that a minimum CSPR can be reached and so leverage this knowledge we can use the dynamic perfusion to screen for the minimum CSPR which show. Show the limitation of the cells and the video combinations in the case of steady state profusion because we control the obesity at a constant level. So as a profusion rate, a constant profusion rate, you may expect the CSPR to stay at a steady level. And using the steady state profusion, we can look for the critical CSPR which is I'll be showing you in the next few slides. So the first applications we use the breeze to screen for the minimum CSPR and from the graph you can see that in the steady state for fusion we allow the cell to reach the maximum level and CSPR is allowed to reach the minimum level and so in the table in the bottom table. You can see that the CSPR is was determined and busy. Maximum busy was as well characterized. And you can see the the minimum CSPR is a highly clone dependent. Some of the clone for example chose chosen B you get to reach 6 people liter per sales per day or some other chosen clone. You cannot go below 19 people liter per sales per day. So this suggesting that. The CSPR minimum is highly con dependent. So the second applications we use the breeze to identify the critical CSPR. So what is critical CSPR which is defined as the lowest CSPR at which the steady state criteria is still maintained. So it means that if you. To perform a profusion culture at a lower critical CSPR, the steady state may be lost. The screening methodology using in identifying the critical CSPR is the push to low approach. So this approach is illustrated in a couple of graphs in this slide. On the left hand side I'm showing the profusion rate and obesity. And on the right hand side is the CSPR space. So if you look into the profusion rate, you see that the push to low approach, we reduce the profusion rate in a stepwise manner and this is reiterated until the steady state is lost. And when you translate it into CSPS space, you can see that the CSPI is also pushed too low in the stepwise manner as well. So the critical CSPR is defined at the. The CSPR as a n -, 1 steps assuming that the status is lost at the end stage. So the here we have a couple of graphs showing that there we generate when we screen for the critical CSPR. You can see that on the left hand side the provision rate will push to a lower level in a multiple step wise manner. And on the right hand side, the VCD was maintained a constant level while the CSPR is also pushed to a low in a stepwise manner until the state is lost. And we quantify this CSPR, critical CSPR and we put that in the table and you also see that similar to the minimum CSPR, the critical CSPR is highly clone dependent and so. This there is meaningful to us in a way that when we design A catalog profusion medium to fit different clone different satellite which have different media demand, we have to balance on the the nutrient needs when we design the cutoff profusion medium. So finally I want to summarize our presentation today by a couple of slides. So first. Now, what should you expect from the Mobius Breeze bio reactors? So the breeze allow you to do intensifications in an easier way. You can capture a high salinity with minimum effort, It doesn't have a complicated accessory and it has a true provision capability. It has a high automation level so you can collect the theater. From high quality data and you can reduce the contaminations and also the human errors, the Breeze help you to save time, save space and save cost. And I want to emphasize here is that the breeze can fix multiple purposes it has. It can be suitable for a wide variety of different applications from the cell line screening. To the media developments, you can rank your clones, specifically proficient clones, using the Breeze. You can perform cellular stability and performance. You can evaluate your media and optimize the raw material and formulations using the Breeze system. And the Breeze, when you combine with other simulation tools or calculation tool, can be a powerful. The kind of powerful tools for predicting cell metabolism. It is suitable for both dynamic and steady state fusions and I can give you an example that if the police can be combined with some of the tool like MVDA and multivariate data analysis and also metabolic flux analysis can be very useful for us to understand the different pathway and different reactions metabolic reaction happening in the intracellular. And so can help us to to be a very solid model. The breeze also highly predictable to the other scales bioreactors. So it can be useful to study different process parameters. You can study profusion and bleed and also the self specific profusion rate that I mentioned earlier and it can help to reproduce the other important condition at a small scales. And it can help you to expedite your organization process. So with that we have the final poll question for you and Mohammed will be going with you very soon. And with that I thank you. I'm happy to take any questions. Go ahead. Thank you along for. So this is the last poll question for which application do you need a scaled down by your actor system? A Clone ranking, B Cell line Stability or performance, C Media evaluation and Development, D Design Space Planning BH temperature DO or E CSPR determination. So I will, as always give you a few seconds to submit your answer. Thank you for your answers. So first, I would like to thank Kevin and Long for this great presentation and it is now time for you to ask you questions, ask us questions. So before we do that, I would like to remind you that it is not too late to send us your question now using the Q&A. WeChat. Please also apply to On Demand viewers. We will try to get through all of them, but if you run out of time, we'll respond to you individually. As a reminder, this webinar will be available on your way on our website soon. All participants will receive an e-mail notification when it is available for viewing. Now we'll start answering your question that have come in. I will. So the first question. Have been asked before the webinar. So the first question is what type of provision cultures and cells compare are compared that this webinar. Yeah, I think I I have, yeah I would like to go back to a few slides earlier that I mentioned that we use this predominantly for the chose cells. Including the chosen choke one and some the G44 clones that we use comparing this webinar. Thank you. So a question that has just been asked. So could you please explain the evaporation correction in a bit more detail? You are adding the eye water to the media, sure. Let me go back to a slide. So evaporation correction happens in two ways actually. The the 1st way is through this volume correction. During batch cultures, we intentionally allow the volume of the reactor to shrink over a set period of time, at which point we actually backfill the reactor back to its nominal working volume by adding the I-1. During perfusion culture, we have steady flow of fluid anyways. So instead of doing that, we actually trickle in DI water with the pump at A at a known rate based upon our previous characterization of the evaporation rates. Thank you, Kevin. And the next question is what is the highest cell density before there would be forming problems? I can take this question. So so he has the highest cell density we could reach up to 350 million cells and because the breed can operate in a bubble free manner, so we don't experience any kind of bubble formation inside a bioweather at all time. OK, thank you. So next one is? What sort of sterilization is used for the consumables? I can take that one. So the consumables are gamma irradiated inside their packaging, so the entire internal surface area of the reactor should be irradiated okay. So the next one is how are bubbles. Avoid it given you are using a highly permeable membrane and active compression expansion expansion, yeah, I'll take that one as well. So that's a good point. There is a additional feature of the system that you did not talk about, which I bring the slide up in the growth chamber. There actually are tiny sections that are constantly pulling vacuum as well. So there is a negative gas removal of a small quantity at all times during the reaction. In addition to that, if we when we do the mixing cycle but and and have intermittent delay where we have stoppage time between each of the mixing cycles, that also helps to give time to remove the gas once the cell density gets high enough. Then this process gets handled by the cells themselves and the bubbles don't won't appear from that either. OK, thank you. So the next question is, so is having only four units in such small scale system seems to be pretty low. Is there any opportunity to extend the system to more by reactors or would a new system then have to be acquired, I can take that one too. So so each system does operate with four reactors, but mostly most of our customers actually purchased multiple units to run more experiments in parallel for exactly this reason. The software does also support live plotting of all the systems in a single interface. So if you're looking at live data that can be viewed from one screen independent of the number of systems that you have, OK, so next question, the next question is? Can you comment on how one can make the shear sensitivity on the cell line from bubbles and impellers? I can take that one too. So we have, we are starting to do some characterization studies on this and there is as we talked before this the reactor can be operated in a way where the cells experience the shear that would be experienced during peristaltic bumping. By by doing that at a fixed rate, you could try to model the damage that you would see from high shoe inside the system. OK, so I will take one last question. So the last one is can you exchange media easily throughout one process? For example, after after 10 days of cultivation, yeah, I can take that question. So so yes, for sure the breeze is viewed with the purpose to help use like easily exchange the media after certain times. If you have a tube welder, you can prepare your media interest in a transfer bottle and you can Weld it into the consumer bottles and use a series to transfer it sterilly. So that's a very easy step to do with the free system. Okay. So, so yeah, So thank you very much for all the questions. So if we did not get to your question, please feel free to e-mail to the presenter directly and I will show you. The contacts of our two presenters, so to register to future webinar and to access or our archived webinar library, please visit our website. I would like to thank again Kevin and Long for today's presentation and thank to our audience for joining us. Have a great day. Thank you. Have a good day. Bye, bye. Thank you. Come. _1734975255674

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