Hi, everyone, and welcome. My name is Veronica Feinberg and I will serve as your moderator today. Thank you for joining us for today's webinar, Unleashing the power of profusion chosen GS host cell line development for intensified processes. As your moderator, it's my role to ensure that we make the most of your time with us. I'm here today with Vincent Velasi. Vincent Velasi is a Scientist 3 on the Expression Systems and Novel Bioprocess Materials team, part of Upstream and Process Materials R&D and Process Solutions. His goal is to use cell line development and engineering techniques to improve the chosen GS host cell line and enhance the efficiency of expressing bio therapeutics. He joined the company in 2015 and has held different positions in manufacturing and R&D. Vince holds an Ms. in Biotechnology from John Hopkins University and ABS in Microbiology and by Biotechnology from Missouri State University. So before I turn things over to our presenter, I'd like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets that you can use. There you can also find our reaction button, indicated by a thumbs up emoji that allows you to give immediate feedback on the presentations, topics, or anything that stands out. All the widges 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 and 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'll be answered later via e-mail. Please know that we do capture all questions and you will also have the opportunity to participate in a couple of quick poll questions throughout the session. I encourage you to take part in these surveys, and if you're watching this webinar on on On Demand, you can still submit poll responses and questions via the Q&A widget. The webinar is being streamed through your computer, so there's no dial in number for the best audio quality. Please make sure your computer, speakers or your headset are turned on and the volume's up so you can hear the three questions within the duration of this one. Looks like we're having just a little bit of technical issues with Veronica. One second please. She's back. There you are, Veronica. OK, all right. Well, I'm actually just going to get started then while Veronica's figuring that out. So welcome everyone to the webinar. Thank you for joining us today. Like she said, my name is Vince Velassie and I am going to be presenting on some modifications that we've made to our selling development workflow with our chosen GS platform so that the clones that we are producing are higher performing and more productive for next generation intensified perfusion processes. So for the agenda today, I'm first going to start with some background in the industry and what upstream process intensification looks like in the industry as well as some strategies and considerations for the upstream space when you're looking at intensification. And then after I lay that groundwork, I'm going to shift into some experimental design, some workflow modifications we've made as well as some key data highlights to end the presentation showcasing some of the performance benefits we've seen with our new profusion selling development workflow slow. So first just getting into upstream process intensification in the industry is a trend. The industry is trending towards a paradigm shift away from Fedbash towards continuous and intensified. If you're not familiar with it, BPOG is an active industry consortium that is really driving this paradigm shift. You can see the Bioform technology road map from BPOG on the slide which is outlining the industry shift towards INTENSIFIED with the market trends, business drivers, manufacturing expectations and key capabilities and innovation required to meet this this transition. These key market trends are pushing the need for a radical step change in the business drivers and the targets required by the industry require massive shifts in technology innovation and approaches for bioprocessing, perfusion and INTENSIFIED are mainstream. The shift to INTENSIFIED can be slow, however it's steadily increasing due to the overwhelming need to produce medicines at reasonable cost across the globe. 1st in class biologics like your blockbusters may not be subject to cost pressures, but products like biosimilars are and new medium demand therapies cannot be justified in your traditional 20,000 liter large scale fed batch facilities. This combined with the increasing number of modalities in the pipeline and the desire to manufacture regionally, smaller facilities are required which can handle flexible and continuous processes. These processes are expected to eventually dominate early stage clinical manufacturing facilities and the time for us as suppliers to prepare for this is now and we are strategically focused to deliver these key enablers through our focused initiatives like Biocontinuum, Intensified upstream Digital, Biopy and Single Use. So what does that mean concretely when you're looking at upstream process intensification? There are really three key levers that we can use in upstream to intensify. The first one is your process speed or faster. So this is how quickly you can go from your C train, your production process to your downstream purification. The second one is increasing your protein titers or higher, trying to just get as much protein as possible out of your upstream process. And finally, the third one is improving product quality or better making sure we're achieving the highest product quality possible with less contamination, aggregates, charge variants, glycosylation patterns, patterns minimize cell debris and insoluble impurities like DNA and problematic Co cell proteins. For the webinar today I'm going to be focusing primarily on higher or how we can get as much protein as possible out of our upstream intensify process and how you can do that through selling, development and modifications to that so that your cell lines are higher performing and higher producing. So there are a few, there are a multitude of strategies that are available in the upstream space for process intensification. And I'm not going to spend a lot of time going into detail on these, but they range from anything from using your high cell density cryo preservation for much larger banks of your of your clone to switching to an M -, 1 perfusion C train and expansion as well as shifting from your fed batch based production process to a perfusion based continuous production and harvest process with much higher cell densities, longer durations and overall increased product tighter and yield. You can also utilize cell culture media formulations that are specifically formulated for cell expansion and your m -, 1 and also specifically formulated for higher production in your perfusion process. And finally again the focus for my talk today is going to be using a perfusion optimized cell line that has phenotypes that maximize productivity in your upstream process. And what's important about this is when you kind of holistically start employing some of these upstream process intensification steps like your perfuse C train, your perfusion based production process as well as cell culture media specifically formulated for intensified. You're making substantial changes to the environment and the stresses that your cell line experiences when you put them in these in this intensified upstream space. And it's important that the cell lines you're producing in selling development are not only able to survive those stresses, but have phenotypes and characteristics that help them thrive in this intensified area. So when you're talking about upstream process intensification, optimizing your selling development workflow is the start of that intensification work. On the slide here, you can see a visual for a mixed population of cells. So for us, this is our Cho GS, our chosen GS host cell line after we've transvected into it a recombinant therapeutic using our our vector. So this is just a standard map being transvected into our host cell line. When you do this, you have a mixed population of millions of cells all with various characteristics, various phenotypes. And it's our job on the cell and development team to take that population of millions of cells and through various screening assays and elimination steps, whittle that number down to one or a handful of very high producing cell lines. But I want to take that one step further and suggest that it's also our job to select for cell lines that have characteristics that help them thrive in the manufacturing process that they're destined for. So I like to imagine within this population, after transfection, a majority of the clones that we produce are these cell lines you can see in brown or in orange there. These are cell lines with undesirable characteristics like no integration or tighter instability and it's our job to eliminate them. But I also like to imagine within this population that there are what we'll call fed batch clones in green. So these are cell lines that have that are high producing but have characteristics that help them thrive in a traditional fed batch process. They produce high volumetric titer in a fed batch, but they commonly do so through a very high growth, low specific productivity or QP phenotype. I also like to imagine within this population that there are what we're called perfusion clones, which you can see in pink. These are cell lines that are high producing but have characteristics that help them thrive in a perfusion process. Most importantly, they have very high specific productivity with a more moderate kind of optimal growth rate that allows them to scale up efficiently and get to the steady state VCD set point, but also not spending so much energy on growth that it's detrimental to your overall product and harvest. They're also ideally metabolically efficient, shear resistant and extended and have stability that's extended compared to our more traditional Fed batch methods. These color schemes are going to stay the same for the rest of the slides where perfusion data is in pink and fed batch days in green and the clones. So you'll see that kind of consistency throughout the slide deck. So we wanted to find out that whether or not we can make modifications to our selling development workflow and preferentially identify these high performing perfusion clones and then test whether or not these perfusion clones outperformed are traditionally derived fed batch clones. When we put them into a Trooperfusion process with higher specific productivity or QP increased tighter volumetric productivity and yield, again focusing on getting as much protein as possible out of your cell line. OK. We have time for a first poll question, but I'm sorry about the technical issues earlier everybody. I didn't get to finish the housekeeping items and the fact that you have a couple poll questions coming up. You can participate and throughout the session, if you're watching this webinar on demand, you can still submit poll responses and questions via the Q&A widget. And this webinar is being streamed through your computer, so there's no dial in number. And for the best audio quality, please make sure your computer speakers are turned on and the volumes up so you can hear the presenters. Lastly, an on demand version of the webinar will be available after and it could be accessed using the same link that was sent to you earlier. And there's for a certification webinar certification. If you'd like to fulfill the criteria of 40 minutes viewing time and completing 3 poll questions, you will receive that at the end of the webinar. So that was the rest of what I had to say in my introduction. I'm sorry about that again. And here's your first poll question on screen. What appointment or tools are you currently using for small scale perfusion, clone screening, media development and process development deep well plate models? TPPP Spin tube models, Amber 15 or Amber 250 single use or STR bioreactors or none of the above. I will give you a few moments to answer here. Just go here and see if we have any movement on the slides 31 to 50 submitted. I think we still have some more people that need to submit. We're going to close out the poll questions in a couple seconds, everyone. And then, Vince, I think you can move it to the next slide when that's all set. All right. Thank you everyone for your participation and the poll question here. So now that I've kind of set the stage for what we were trying to accomplish and kind of the the reason and the background for it, I'm now going to shift into doing more experimental design and how we were trying to answer these questions. So to address some of the questions that I just posed, we decided to perform 2 selling development work flows head to head, taking the clones that are derived from each and then seeing what differences in growth and productivity were present in those two different sets of clones. So we took our more traditional fed batch selling development workflow and performed in parallel with a new and optimized perfusion CLD workflow to see what difference is in in in clone performance. At the end of the day, we would get. So on the slide here today you can see that our selling development process from transfection all the way to the MINI cool stage. On the top is our more traditional Fedbash based selling development workflow. And on the bottom is our new perfusion selling development workflow that more or better reflects the perfusion process with the right media and scale down assays and clone characteristics. So we started with our chosen GS knockout wholesale line and we transfected into it a pretty standard to express IgG. After that transfection we played it out many pools in our Salvanto Forto C cloning medium and static, allowing those many pools to recover from GS selection and become confluent. At this point we took all of those many pools, they were in static and we screened them with a seven day static productivity assay ranking them from highest to lowest tighter. We then scaled up the Top 40 mini pools into suspension cultures into our CD fusion growth media and split them into two distinct biological replicates. SO40 mini pools biological replicate A which was sent down the Fed the Fed batch selling development workflow and then the other replicate or replicate B of those 40 mini pools was sent down to profusion CLD workflow. So on the Fed batch selling development side, we took those 40 mini pools first, screen them in a batch essay looking at growth in productivity data using our advanced show fed batch medium. We then further characterized a 24 of them in a scaled down TPP Fed batch assay looking at Fed batch specific growth in productivity data using our advanced show fed batch medium and our Silvento modified prime. Using that data we identified the top five Fedbach mini pools for cloning on the perfusion selling development side of this project. We took those 40 mini pools, they were in suspension. We first screened them in ATP batch essay using our advanced HD perfusion production medium this time and then we further characterized 24 of them in a 24 depot plate simulated perfusion assay looking at perfusion specific growth and productivity data. And this scale down assay is just our to the best of our ability US reproducing the perfusion process in a very cost effective and high throughput manner. So using that assay we identified our top five perfusion mini pools for single cell cloning and then we moved on to the next stage of the selling development process which is the cloning stage. We took our top five Fedbash mini pools and top five Perfusion mini pools single cell, clone them into our Fortune C cloning media using facts and allow those clones to recover and become fluent. We then screen them again with a seven day static assay ranking them from highest to lowest titer. On the Fed batch selling development side we took the top 80 fed batch clones in static, scaled them up into 96 depot plate suspension cultures in our CD Fusion growth media. We then screen them with a high throughput depot plate Fed batch assay again with our Fed batch medium and feed looking at Fed batch specific growth productivity data and ranking them from highest to lowest performance. The top 12 Fedbash clones were identified in this essay scaled up to TPP for you know further characterization and banking on the perfusion CLD side, we took our top 80 perfusion clones that weren't static, scaled them up to 96 depot plate suspension cultures. We then went through an adaptation step where we took all these clones and adapted them to salvento 42X. This is a expansion media that is specifically formulated to 8 in your N -1 expansion before going to your perfusion process. And while it's not a requirement for adapting going into your perfusion process, there are potential benefits to growth and overall productivity when you do this. So we chose to implement this adaptation step at this stage in the selling development process. After that adaptation was complete, we did a 96 well version of our simulated perfusion assay, ranking these clones from highest to lowest performance based on first and foremost their QP, followed by their volumetric productivity, while making sure that they had some minimum growth requirements. So using that data holistically, we identified our top 10 perfusion clones, scaled them up to TPP for further characterization and banking. So at this point we had done these two selling development work flows in parallel, producing 12 top Fed bash clones and 10 top perfusion clones, and we were in a position to start ask or answering the questions we asked at the beginning. So we began to put these clones, these two sets of clones head to head and some of our scale down assays. So this is the TPP Fed batch assay as well as the 24 depot play perfusion assay. And we also took the top clones from each selling development process and evaluated them using the Mobius Breeze microbioreactor and a true perfusion process. Again, just trying to answer those questions that we posed at the beginning and find out if the perfusion selling development clones that we produced outperformed our traditionally derived Fed batch selling development clones when we put them in a true perfusion process with higher QP increased tighter volumetric productivity and yield. Again just with the focus on getting as much protein as possible out of your cell line for intensified. With that, I'll pause and give it back to Veronica for the next poll question. Thanks, Vince. So you'll now notice your next poll question on the screen. What clone characteristics do you find are presenting the most significant challenges in manufacturing scale perfusion processes growth? So low VCD and IVCC, high CSPR or low EP, high oxygen demand or poor product quality give you about 1520 seconds to answer here. Let's see, we're almost at 40%. Do you have any more answers? Almost at 50. Thanks everyone, for submitting. It's going up still. OK. Yeah, keep going. More votes. We're going to close out in a couple of seconds. Everybody. Vince, whenever you'd like to move this slide over. I think it looks like a good amount unanswered. OK. Yeah. Thank you again everybody for your participation in the poll question. So now that I've kind of gone through the background and what we were trying to accomplish and also the methods for how we approach this with the time we have remaining, I want to give some key data highlights to the output of this project here. So the first part in this process where I'm going to show you some highlights is this 24 depot plate simulated for Fusion assay. So yeah, so we again, we took our top 12 fed batch clones and our top 10 for fusion clones. So there's 22 clones in total. All the Fed batch clones were being passage and expanded in our CD Fusion Media which is just like our standard growth media we offer. And the Perfusion clones were being passage and expanded in that cell into a Fortune X expansion media. We then seeded each clone at 10 million cells per mil and three mils of our HD Perfusion production media and that 24 depot plate. We performed daily half DVD media exchanges which is replicating the Perfusion process in a semi continuous fashion while looking at growth productivity metabolite data daily for five days. For the next few slides I'm going to show you the average clone data from each set of clones and and that 24 well assay. So the first thing we're going to look at is growth, so VCD viability and cell size and right away you can see the Perfusion clones grew slower, reached lower BC DS and were slightly larger than their fed batch counterparts. With the perfusion clone average, we can see in pink on the BCD graph reaching just under 40 million cells per male in this assay, while the Fed batch group reached closer to 55,000,000 cells on average, Both sets of clones maintaining very high viability throughout the essay. When you look at the cell size, which you can see on the right again, the perfusion clones were 5 to 10% larger on average than their Fibesh counterparts. So right away we noticed a distinct difference in the growth phenotype between these two sets of clones. When we looked at our tighter or volume measure productivity that was produced, we found that the perfusion clones on average produced higher tighter than their Fibesh counterparts. We collected the tighter throughout the essay and on day five or the peak tighter day the perfusion clones produce close to 2G per liter while the Fed batch clones were just under 1.8. There was an 11% increase in tighter in the perfusion group compared to our fed batch or more traditionally derived clones when we looked at the total yield that was produced and collected during the scale down assay. So to calculate this, we took the 1.5 mills of supernatant that was collected each day during the media resuspension, multiplied that by the tighter that we measured and then added that up throughout the five day assay giving us the total yield or total protein collected for each clone. And then we took the average for the perfusion and the Fed batch group. Again, we found that the perfusion group on average produced 16% higher yield that was collected and compared to the Fed batch counterpart. And again, we're focusing on trying to get as much protein as possible out of our cell lines. And this is an area where the, the perfusion group of clones is outperforming their fed batch counterparts. And finally, looking at specific productivity or QP, we found that the perfusion clones had substantially higher specific productivity than the Fed batch clones. And again, we know this is a key driver of overall perfusion performance. It's also one of the areas where we had the biggest impact and the biggest change in performance with the selling development modification. On peak days, the Perfusion clones had 45% higher QP than the Fed batch group, which is a pretty large increase. We also ranked all the clones in this assay by volumetric productivity yield QP as well as their VCD Max. Again, perfusion in pink and Fed batch in green. And when you look at those key productivity characteristics, you see that the Perfusion clones are for the most part outranking all of the Fed batch clones. The trend is reversed when you look at the VCD Max where the Fed batch clones are growing more and the Perfusion clones are not. So what we took away from this kind of ranking shift is that effectively what we've done by changing our selling development process, it selects for clones that are spending more energy on productivity and less on growth, shuttling more energy towards recombinant therapeutics and less towards towards growth, which overall is is leading to a more productive cell line, a more productive process. So if you combine those rankings with the increases in tighter yield QP, really the take home message at least in the scale down was that with the changes we made to our perfusion COD workflow we saw an improvement in productivity compared to our more traditional selling development methods. However the scale down assays like the 24 depot plate that we use, these are excellent high throughput tools for identifying your top clone candidates. So for us this is going from like 80 to 100 clones down to 5 or 10 but they lack the scalability and process control of a true perfusion system which makes it difficult to predict your actual perfusion performance, make final clone decisions. So going from like 5 to 10 clones down to 1 lead lead clone candidate and also to begin your process development. So this is where the Mobius Breeze microbioreactor comes in, which if you're not familiar with it, it's a two mil automated single use perfusion cell culture platform. It is really useful for for perfusion selling development and clone screening like I'm showcasing here in this webinar but also useful for media screening and optimization as well as early process development. On the slide here, this table showcases that in a little more detail. So you can see kind of the working conditions of the 24 depot plate semi continuous perfusion system we use alongside the Mobius Breeze as well as your larger scale bench top perfusion reactors. And right away looking at this table you can see that the Mobius Breeze is much more representative of a Trooper fusion process with much more process control and is much more similar to what you'll see in your scale up or larger perfusion processes making it a better model for screening and a better representation for overall clone performance in perfusion. So to validate what we saw in the 24 well assay we we repeated that head to head evaluation taking our top clones for me so in development workflow and putting them head to head in the Mobius Breeze with A1 DVD dynamic perfusion process for 11 days. I want to drive home that we used a very standard templated dynamic process with no clone specific media development and optimization and no clone specific process development. This is a very bare bones process, just really looking at the baseline performance of the cell lines and making sure that it's the date is comparable. So looking again at the growth first, similar to what we saw in the 24 well our perfusion clones reached lower peak PC DS with very similar viabilities compared to their Fed Bash counterparts. You can see the peak PCD graph in the middle there, with the Fed Bash clones on average reaching close to 130 million 135 million cells per mill, while the Perfusion clones were just under it, 100 million cells per mill. So there was a 26.2% decrease in the peak BCD and the perfusion clone group on the left. You can see the individual growth curves and BCD curves for each clone and I have this in here just to showcase that. Although there was a significant difference in the peak BCD that was reached, there's still a wide diversity of clone growth phenotypes in these in the set of clones with some clones reaching, you know 50 million cells per mill peak and others reaching 200 million cells per mil. And across the board all these clones were able to reach their peak PC DS and maintain it quite well throughout this 11 day dynamic process while maintaining very high viability with almost no differences between the two sets of clones and viability. When we looked at the productivity characteristics, again similar to what we saw in the 24, well our perfusion clones produced more than the Feb bash counterparts. So looking first at specific productivity in the far left there QP whether we looked at the average QP or the peak QP that was reached, our perfusion clones produced approximately 60% higher QP, so an even bigger increase in QP compared to what we saw in the scale down. And I also want to point out that some of the top performing or highest producing cell lines on the perfusion side we're hitting QPS at of 30 and above when you look at the volumetric productivity in the in the middle. So this is just tighter in micrograms per mil per day that was being produced. Whether you look at the average volumetric productivity or some of the peak days, the perfusion clones were producing approximately 20% higher volume metric productivity averaging around 1.3g per liter per day, with peak days reaching closer to 1.5g per liter per day. Not surprisingly, this 20% increase in volume metric productivity translated into a 22% increase in overall total yield in the perfusion group as well, with some of the top performers in the perfusion group hitting closer to 50 to 75% higher total yield than the Fed batch average. Again, the whole purpose of doing this is to see if we can really squeeze every ounce of productivity out of our cell lines and do so through cell line selection. So even though we gave all of these clones the exact same amount of nutrients, the same process and everything, the perfusion clones were more efficiently turning that into greater yield, which at the end of the day is our goal for upstream process intensification on this slide. This is the same data that was shown on the last couple slides. So you have the peak VCD on the X axis in the peak QP for each clone on the Y axis. I like this graph because it showcases the same data, but does so on a single graph. You can see right away the Fed best clones in green are primarily falling down that bottom right quadrant of the graph which with a couple outliers, while the perfusion clones are spread more towards the middle and the top left of the graph. This graph is really informative because when you look at it in the bottom left quadrant, these are cell lines or clones that have very low specific productivity and low growth or low VCD. And we typically try and avoid this especially as you get down to the axis in the bottom right of this graph, this 4th quadrant. These are cell lines that have very low specific productivity with high growth. And while they might, might do OK, it's very difficult to increase your cell lines QP with media and process development so that it's it's harder to improve the overall productivity of these cell lines in this top left quadrant or quadrant one. These are clones that have very high QP with low growth or lower VCDQP is very tied to your clone phenotype and you have a better time increasing VCD with your media optimization and process development, meaning they're in a better position to go forward into the next stage of your intensify process development in the top right quadrant or quadrant 2. This is your kind of Goldilocks down or ideal quadrant you want to be in with cell lines that have very high QP with high VCD as well. You can see that naturally fresh out of the selling development workflow, none of these clones are really falling up in this top right quadrant and it's the goal of media and process development to take your clone from whatever starting point you're at and push them farther and farther up into this top right quadrant. The take home message from me for this is that by making the changes we made in the selling development workflow, we effectively shifted this kind of balance of growth and productivity towards a more more productive state. Meaning the clones that are coming out of our perfusion selling development process and workflow are in a better starting point to begin media optimization process development. And hopefully that translates into a much more productive perfusion process. So because we made changes to our selling development workflow and we wanted, we wanted to make sure that the clones we're producing are still stable since stability is such a big, big part of selling development and clone selection, but especially for perfusion and intensified. But just like we required a more holistic and expanded understanding of what makes a clone high performing in our perfusion selling development workflow. If your clone is destined for a continuous steady state perfusion process, it also requires a careful assessment of your selling stability. So unlike what we've historically done for stability where we take our sellings out for 20 passages or 60 PDL approximately, we doubled that time and increase our stability passaging time to 120 PDL or 40 passages before evaluating these clones. And we also expanded our stability criteria to not only include tighter like we would do normally, but also QP and BCD are growth. And what you can see when you look at this stability graph for each of these clones that across the board, they're all very stable despite the changes we've made. But I want to draw attention to some nuance here where you have cell lines like B4 and B6 that are maintaining around 100% productivity, like they're tighter after 40 passages, but they're also maintaining a very stable QP and VCD as well versus cell lines like a seven. That even though it has 100% tighter after 40 passages, you can see there's a shift in your QP in your growth. That phenotype is kind of shifting away from a high QP to a higher growth phenotype. This may not necessarily matter for your more traditional Fed batch process, but if you're talking about doing a very long potentially 90 day perfusion run, having a cell line that's able to maintain that balance of growth and productivity along with this tighter may be beneficial for your perfusion bioreactor operators and give them an easier time establishing and maintaining a long steady state. So I think with that that's really all that I have here to to share here today. But my take on message is that if you're doing upstream process intensification, optimizing your selling development process is the start of this intensification work. We showed here that a perfusion specific selling development process can lead to higher producing clones for intensified. Our perfusion selling development clones outperformed our Fedbash clones and both a 24 depot plate high throughput assay as well as the Mobius Breeze microbioreactor with higher QP tighter and yield. And I guess really the the take home messages regardless of which cell line, which platform you're using to produce your therapeutic, your selling development process should mimic your desired manufacturing process to the best of your ability. I think it's common to take cell lines from a fed batch process and just repurpose them for perfusion or vice versa. And while this may work because of high producing cell line typically is high producing, you may be leaving some production and and some performance on the table by not replicating your process during selling development. I think with that, I'll pause and give it back to Veronica for the last poll question. Thanks, Vince. Thanks for this great presentation. So before I read this poll question, I just wanted to let you know, we do have AQ and A coming up. So this is our last poll question. Then right after this we'll jump into that. So our our last poll question, are you interested in evaluating the Mobius Breeze microbioreactor at your site, yes or no? Few more moments to answer. We're getting up there. We're almost at 50% of attendees answering, which is great. Awesome. We're over 51%. OK, we're going to close the slide in a few moments. If everyone can make your selection and do you want to advance it whenever you're ready then. Breeze Popular, like Veronica said, we will have AQ and A following this. So don't go anywhere. But before I before we get there, I do just want to give some acknowledgements to the team here and just thank everyone that was involved in generating all the data that I shared here today. Nothing happens in a silo. So a big thank you to everyone here listed on the slide. And with that, I think we can move on to the Q&A, Veronica. So I'll give it back to you. Yeah. OK, so now it's time for the Q&A section of our webinar. But before we start, I'd like to remind you it's not too late to send us your questions now using the Q&A widget. This also applies to on demand viewers. We'll try to get through all of them, but if we run out of time, we'll respond to you individually. And as a reminder, this webinar will be available on our website soon and all participants will receive an e-mail notification when it's available for viewing. OK. And now we'll start looking at the questions that came in. So I will I'll read the questions and Vince we'll we'll answer these. So let me see the first question. The Breeze is only A2 milliliter culture volume. How can you get enough sample to get the needed growth, productivity and product quality information needed? That's an excellent question. So the breeze is A2 mil working volume and when you sample for offline BCD measurements, we take approximately 140 micro liters out of the breeze and that's used for an offline measurement, something like a bicel giving us our our cell counts. And then you're still collecting A1 BBD harvest every day, at least in this experiment, sometimes even more depending on your process. So we collect one mil of perfusate or harvest each day and that one mill is more than enough for your product quality analysis as well as your tighter analysis. Thank you, Vince. Another question from the audience, are there any differences in glycosylation pattern between conventional Chow or GS system and this one? That's a great question. So I don't have that data present in the slides. And actually that's a good reminder that I just want to tell the audience that in addition to this webinar, we're working on submitting A manuscript as well. So keep an eye out for that. And within that manuscript, there is some supplemental data that includes all of the product quality we did. But to answer the question, while there were some minor differences in product quality and glycosylation, there's nothing that's concerning, nothing that would wouldn't be addressed with your next step with your media, with your process as well. So no concerning differences in product quality between these two selling development work flows, but we did do that. I'll just keep an eye out for the OR for a publication as well to see that in more detail. Thank you. OK, our next question, did you optimize the semi continuous feeding to mimic perfusion? Was changing 50% the medium daily the most representative way to mimic a real continuous perfusion process? Yeah, we've done quite a bit of work optimizing those scale down models, whether it's the the 96 well or the 24 well semi continuous perfusion model. And the answer to the question is yes, at a .5 DVD or a partial DVD. We see that that's more representative of what you'll see in the actual perfusion process when you do a full DVD. It although it's maybe a little easier on you to do that. You're taking out some of the byproducts that are sitting in there like lactate, the things that are in the media all the time in a true perfusion process. And whenever you do that, it's a little less representative. So it's yeah, it's better to do a half BBD. It's just more representative of growth and productivity. Another one we have here. Can you expand on what Perfusion specific clone characteristics were used to select your top Perfusion clones? Yeah, So we. So first and foremost we ranked all of our clones by QP. So we we took a short list basically of any clone that had a QP greater of 30 and that 96 well perfusion essay and then after we took the clone they reached at least 30 for their specific productivity. We then ranked them by their volumetric productivity from highest to lowest, and also made sure they had some minimum doubling time, maintaining high viability of some of the clones that didn't grow as well but had QPS that were very high had lower viability and slightly too long of a doubling time. So they either naturally got weeded out during scale up where they couldn't survive the scale of process, or we eliminated them just based on, you know, too long of a doubling time with viabilities at like 95 or lower percent. In the Mobius Breeze, bioreactor be utilized to evaluate the sheer force resistance of those clones. It's a good question. So this is actually something that we've been looking at our team as well. So shear resistance in your cell lines as well as ways to model that, whether it's in your scale down assays or in the Mobius Breeze. And I think there's some potential there to modify the shear levels within the breeze so that it can represent a high shear environment. But it's just something that we're still looking at. It's not something that definitively. I can say yes or no, but keep an eye out. Maybe that's a new webinar in the future. I don't know, OK. Do you anticipate any new challenges in perfusion process development with these high QP clones? Yeah. So what we've effectively done by changing our selling development workflow and producing these very high QP clones is we've also increased maybe their nutrient oxygen demand because not surprisingly if you have cell lines that are very high specific productivity, they require more energy on average than maybe your your lower QP clone. So there is potential that by shifting towards a high QP phenotype, which is really one of the goals we had for for next generation or continuous on the selling development side, you may increase the burden on your hardware and on your process to deliver enough nutrients in the media as well as main sure making sure that you're oxygenating the the bioreactor. So there are some potential next steps or challenges that we can address to minimize those Those things. I want to say that maybe we also have a crisper screen for for our Cho cells that is available I think as well now. And it's a really good tool that we have that could potentially be used to address some of those challenges as sort of a next steps for this project if we run into some additional challenges when you scale these up into larger bioreactors. Next question would be what about cost effective for perfusion versus fed batch? Cost effective, Can you read? That like I think it's what about cost effectiveness for perfusion versus fed batch let's. See. Yeah, can we maybe whoever submitted that elaborate on that just a little bit more to, I'll do my best to answer the question, but while they're doing this the next one. Yeah, I can go to the next one. Let me see are are we typically try to minimize the number of media we use? Here you have 3 distinct media formulations. What's the advantage of using multiple media? Yeah, that's a great question. So I think they're probably referring mostly to the expansion media in this instance. So with the expansion media that we used here, there's not a requirement to utilize it. So you can expand infusion, you can grow infusion and still go into your intensified to say you only have two media there. But we've seen with some of the clones we have, if you do use expansion media just because it's more similar similarly formulated to our perfusion production media that there are some added benefits. We see some cell lines that grow a little better and can reach higher VC DS or maintain higher VC DS in a perfusion process with slightly higher QPS as well. And it's added benefit of a slight increase in growth along with a slight bump in your QP leads to an overall more productive system. So that even though it's an extra media that you're implementing early, early in the cell and development process, doing so can lead to some added benefits and performance benefits when you get to your manufacturing run. Thank you. And then next question, what resource investment is needed to run the Breeze bioreactor and what kind of throughput can you get per FTE? So I think currently with the way the Breeze is, I want to say that a full time FTE that's running a standard process on there can get around 12:00 or so pods run at a time, maybe more. But I think yeah, 1212 is probably fair and I think there's some improvements to the software and to data management hopefully that will be implemented that'll increase that number. But right now I want to say it's safely in FTE. You could do 12 or so pods, so or reactor or reactor vessels, everyone I'm starting. OK. And then I think we have, I think that was the last question there. If anyone else has any other questions, you could feel free to submit now or you could we'll answer your questions after this webinar individually. Yep, I believe we answered the last one is are you going to do a webinar on acceleration or N -1 perfusion and intensified fed batch that's. A great question, Veronica. Do you know if we have a webinar already on that available on demand? That's what I was thinking too. It sounds familiar. Let me look into that and whoever asked that question, we can get back to them on that. So that concludes all the questions we don't have, We don't have any other questions coming in. So I guess we're going to close it off a little early today. Thank you everybody. We didn't get to you. Please feel free to e-mail Vince directly to register for future webinars or to access our archived, archived webinar library. Please visit our website. I'd like to thank Vince for today's presentation again, and thanks to our audience for joining us. Have a good day everyone. Thank you very much for attending. Have a good one. Bye everybody. Bye, bye. _1734975255623

     

   

       

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