Our panelists, three prominent women in science, went from concept to custom media formulation to data in just over a month. Learn how you can design media in minutes with NB-AIR™ that performs better than off-the-shelf media formulations. In ONE MONTH you can progress from CONFIGURING custom formulations to reviewing proliferation RESULTS.

With NB-AIR™ simplify and speed the formulation process to improve CONSISTENCY, QUALITY, and SCALABILITY thus expediting your research and development from the bench to bioreactor. The panelists are:

  1. Saba Ghassemi, PhD, UPenn
  2. Lori Noffsinger, Cognate Bioservices
  3. Asma Ayari, PhD, Nucleus Biologics

Webinar Audio Recording:

Listen to the audio:

Hi everyone. Thank you for joining today. I see folks are still rolling in so we'll wait a couple of minutes to dive into the content, but thanks for joining us.

All right, so thank you for joining us today everyone. My name is Rachel Gauvin. I’m Head of Human Potential and Program Management here at Nucleus Biologics and we are thrilled to have you here for our Path to Media Mastery, Part 2 webinar today. So, we'll have some great content to share, some great data. I encourage you to use the chat and the Q&A boxes throughout the webinar to send comments and questions. We'll have a great session at the end to address all of those, but without further ado, we can get started, so Brad...

All right, hello everyone, good morning uh good afternoon I suppose as well to many of you. Just jump right in as Rachel said to talk first and foremost before I make the introductions as to why we're here. We're here to see some really really compelling data that was produced using formulations that we, I should say the scientists, generated live online just a little over a month ago, not too long ago, so that's the exciting piece of this.

They were able to empath media mastery using NB-AIR to generate formulations live as you guys saw, and we were able to deliver, implement, perform those studies, and now we even have data to show you guys today which is all a really really exciting turnaround to be able to do. So, I will make the introductions for those of you who were not able to join and these are in kind of order of, in the speaking order, so we'll start off with Asma Ayari who is a scientist here at Nucleus Biologics, Saba Ghassemi, who is a Research Associate Professor at UPENN Center for Cellular Immunotherapies, Lori Noffsinger who is in Operations and Development at Cognate BioServices, and I’m kind of new to the screen, my name is Brad Taylor, Vice President of Marketing at Nucleus Biologics.

Myths About Custom Media

And just for those who are kind of new to the topic, new to maybe to Nucleus, we want to do a little bit of a rollback and kind of really talk about some of the things that we find so exciting about custom media. Why we think it can be transformative, why we think it's really really important to our industry as a whole. So let's talk about some of the myths that are floating around out there. 

Firstly, the specifics of your formulation aren't really important, and I’m actually going to call to Saba to talk a little bit about this, but we've been able to show in publications and in the publication history that even single component changes can really improve things like transfection efficiency, cytotoxicity, yield, potency as we'll see in the next slide. This was some fantastic work that was done in conjunction with UPENN looking at CAR-T cells in a xenograft model of glioblastoma and Saba, if you want to since you're the first author and we have the privilege of having you on the call, I would like to see if you want to say a few words to this.

Sure. Hi everyone. So, this is a work that we published last year in collaboration with Nucleus Biologics, and we, as Brad mentioned, we realized that there was an underappreciated role of components in the media and if you change components, that would enhance and affects cell therapies, so here, for example, we showed that if we change one factor and then here we replace serum with Physiologix, which is basically concentrated growth factors, it enhanced the potency of CAR-T cells in xenograft model of neuroblastoma actually.

We had mice and we injected tumor cells into these mice; and we had two groups that we compared with each other. One group was CAR T cells that are expanded in media that contain serum, and then one group of cells that are expanded in media that contained Physiologix. And we saw that there is a dramatic impact in the efficacy of CAR T cells - we had done a stress test, we changed the dose of the T cells and we saw even though in the lower dose, CAR T cells that are expanded in Physiologix, they clear the tumors better than their counter group, so that basically told us that changing only one component for this study made a dramatic impact.

Alright, thank you Saba for walking us through that. That is an extremely important point as well especially when you take into account the fact that many media providers out there don't truly let you know what's in their media right? So, we need to really take account as to what we're adding or depleting and how we're manipulating that media, and that's certainly why custom media to us is, again, something that's very exciting. It can be very very transformative because if we can use that media to increase toxicity and potency, then that's certainly a game changer for us.

Alright, another myth - researching and configuration is time-consuming. Well, this one, I’m gonna give it to you. It can be if you look at the next slide, you'll see that there is just simply a mountain of data out there. This is data taken directly from PubMed. It's the number of T-cell publications that are currently available on PubMed and you can see that number there, which is kind of awe-inspiring just for T cells, 512,299 T-cell publications historically, this is increasing steadily every year as we all know there's a lot of research being put into T-cells, you know CAR T-cell therapies as Saba is working on and others, is increasing and is very exciting.

So you know, we anticipate this to only increase. So how do you really quickly dive into those publications and find those components or those pieces of data that give you indications how those components are going to affect the outcome of your study, and when you find a bit of information, how do you really determine is this really the best information? So, in other words, how do you canvas all this data? How do you canvas all this information quickly and in an expedited fashion in order to get you guys to your studies the quickest? Well, the answer is as we're going to show you guys today, and have shown in the past.

Traditional Custom Media Production

NB-Lux was kind of one step in that direction, by shortening the configuration, the ordering time, and the manufacturing time. But NB-AIR has really tackled another aspect of the timelines that it takes to produce custom media. We can kind of start off with your traditional overview of how custom media was produced and it is time-consuming and a tedious process. It begins with a lot of research, as you see, and with the blue area of that bar moving into configuration and quoting, which is a manual process, which again can be very time-consuming.

Manufacturing, because it's typically done on a large scale, can take a number of weeks to really produce and deliver. And then, of course, you have to do your testing. This is typically repeated because typically there's a second round where you want to tweak something within that formulation - you want to add or increase or perhaps decrease or change something about that formulation to make it even more efficacious. If you hit next, we will see the comparison to proprietary-off-the-shelf. As the name implies, of course, it's off-the-shelf, so the manufacturing has actually been done prior to it being put on that shelf so, of course, your manufacturing time actually becomes a little bit more of an order and delivery time.

Research stays the same, but you can see the time to actually starting to implement and utilize that formulation in your studies is dramatically decreased to somewhere around, I believe, 23 weeks. Now what we've been able to do at Nucleus Biologics, if you hit next Rachel, actually compares as far as the timing to what you see proprietary off-the-shelf. We've been able to shrink that ordering time, that customization, that configuration time by using online tools to expedite the process and now, today, we're taking it one step further with a combination of NB-Lux and NB-AIR that further shrinks the research time taken in order to produce that custom media.

At the bottom, we have a complete breakdown of the number of days that are involved and you can see if we summarize the days, it's only about 144 days or around 20 weeks, 20.6 weeks to move from formulation to your final media formulation in the laboratory compared to traditional custom which comes in at over a year to see those same results. And then again, you know, with the introduction of NB-AIR, we're saving somewhere between two to three weeks off of your current timelines. Let's go on to the next one.

Alright -  I can't afford it. This is another one we like to hit head-on at Nucleus. We can scale with you, we can offer volumes anywhere between 2 liters and 2,000 liters. We have very low minimal order quantities, so like I said, you can order two liters and sometimes it does come with a bit of a price, especially at the small liters, but once you start to move to volume, once you really start to scale your process and scale your studies, you're really going to realize a lot of cost savings actually. We did a case study in the next slide. If you're just looking at a case study with high commercial volume around 5,000 patients a year, estimating somewhere around 14 liters per patient; if you compare the proprietary off-the-shelf, and this is just an average taken of the media and all the components that one would need to purchase and add to that media, it's somewhere around $300.

Custom media formulation at those volumes is only going to be with a similar configuration as what you're seeing with the proprietary, it's only $189. This equates to around $13 million a year, and a delta of $80 million between what you're seeing with the custom media and the proprietary, not to mention the fact that with custom media you own your formulation, you only have one vendor to qualify, and you only have one process to validate versus the proprietary off-the-shelf. You don't own that formulation, you don't know what's going in it, you don't control the supply, you have multiple vendors, products, and processes to validate and qualify.

And then finally, you know, I mentioned ownership there you know that's very very important aspect. Ownership, transparency and then the consistency of supply, such a huge issue right now in our industry. If you look at the next slide, COVID-19 has added another level of complexity to the already kind of difficult supply chain and consistency that the cell and gene industry was already seeing.

So you're seeing shortages of supply from cell culture media to reagents and you see this in the news articles, in the headlines, you know from the scientists to other business journals and you have to be cognizant of are you going to be able to source all that material, and if you're sourcing different components to go in your proprietary off-the-shelf, then all of that is on your plate versus with our model, you're going to one source for these materials and we give you a very transparent lead time in order to receive those materials. So I guess to summarize and then really get to the data, which I know this is what everybody is really here for today, I would challenge that "I can't afford it" should become "you can't afford not to". 

Asma Recap

So, at this point, I'll turn it over to Asma.  We're going to start with Asma, and she is going to do a little bit of a recap for those who may not have seen the prior webinar just to kind of give you a feel for what NB-AR looks like.  So Asma, I'll turn it over to you.

I think you're on mute Asma.

Sorry, thank you Brad, thank you everyone for joining us. I will start by a quick recapitulation of the main criteria that we selected through NB-AIR last webinar. So the cell type I selected to work on was a T cell and then for the CQAs, I selected, it was to increase viability, increase proliferation, maintain cytotoxicity, and the components of interest were sodium selenite, human serum albumin, Physiologix, and l-aladin, l-glutamine. So we're going through NB-AIR to show you how we did that so, and how fast we can set and develop a media to test. So Rachel, if you can allow me to share my screen please. Thank you.

Alright, so here, I’m on the platform on NB-AIR and just a quick reminder, I selected OpTmizer last time and then for the supplement, I selected human serum, and then for the cell type, it's T cells. So when I hit T cells here, it shows all the data available on t cells that we have. So, if I go next, then here at this level I’m able to select the CQAs that I’m interested in.

So my first CQA that I would like to rank as one will be proliferation. And I have the possibility to either select decrease, maintain, or increase. So I’m going to select increase and then the second CQA for me is viability. So I would like also to increase viability. And then the third CQA is cytotoxicity, which is a CQA that I would like to maintain, so after selecting my CQA, I just go next and then at this level, I’m able to select the basal media that I would like to use for my media; and I will go with what Nucleus recommends and then from here, I have all the components that are involved in improving proliferation, maintaining cytotoxicity, and improving viability.

So what I selected last time was sodium selenite, so that's what I wanted to test, and then for the, I didn't select uh yeah so for the amino acid and dipeptides, I selected l-alanine, l-glutamine, or what it's known by, GlutaMAX, and for the protein source, I selected human serum albumin, and then Physiologix. Alright, so then if I hit next here, I will have here the cell type that I selected, the CQAs that I selected with the levels that I wanted to reach, and then if I go here, I see I have the details of the formula that I selected here in amino acid, vitamins, inorganic salt, and carbohydrates. And you see here we have also the possibility to show the value in either milligram per liter or in millimolar.

So, for my experiment that I chose to run last time, you see here I have sodium selenite, so the point was to test sodium selenite so sodium selenite will be the variable component. So I will have zero here for this formula and then I would like to use a little higher level of human serum albumin and then higher level of Physiologix and then L-alanine, I mean I will go with a level that is recommended here by NB-AIR, which is 434.4. So this is the first control without selenite and remember, if I wanted to test this using any off-the-shelf media, I wouldn't be able to do it because sodium selenite is part of IMDM, so if I wanted to test the media without sodium selenite, it would have been a big hassle to have access to this kind of media.

So the level recommended here by NB-AIR is to give an estimate of what the level that you would choose, so what I wanted to do here is to try different levels, so I have low level and then medium level, and then a high level so I will choose like a low level 0.017 milligram, and then I will keep here this one because this is a component that I don't want it to change in my media. And then I will have this one also at 20 and then this will be the second level so the medium level of sodium selenite, and here I will have the highest level which is 1.7 milligram per liter; and then I will have here 20.

Alright, okay, so then I will select these four formula so one, two, three, and four, so here I have all the formula that I selected and then I will save it to NB-Lux and this will take me automatically to NB-Lux and from there, I will be able to place my order; so it takes a little bit time to upload all the formula to NB-Lux. Alright, so here I’m on my account where I have all my formula saved, so if I go on one formula and hit view formula and then load the formula into the configurator, you can see here that I have I have some boxes to check here so the first one will allow me to select what type of media I want if I wanted liquid or powder, so as I want it liquid it will be okay. I’m not going to change anything here - I don't want it to be cGMP and I’m not required, any tested as it's still on a small-scale test, and then for the quantity I don't need 20 liters.

I actually need only two liter and I want it in a bottle and then one liter bottle is okay. So then I just want to check and to show you that the changes I made in NB-AIR were translated to NB-Lux so as you can see here the level of ln alanine l-glutamine was brought here to NB-Lux, so this was for the amino acids and then for the inorganic salts; for example, if I go here and look for sodium selenite, so sodium selenite is over here and so you see I have the highest level that I selected here so I can confirm here and then place my order, and to place the order is really simple - just enter the address and all the information and then confirm your order, and then after two weeks, I received the media that I’m going to show you the data for which I tested the media. So order successful. So Rachel I’m gonna give you back the screen.


Alright, so this is the protocol that I followed for this experiment. So I have the control media which is OpTmizer supplemented with five percent human serum and then I use the NB-AIR suggestion so I have no selenium, low selenium, medium selenium, and high selenium with all of them supplemented with two percent Physiologix. So, on day zero, I activated the cells using dynabeads at the ratio of three to one, and then on day three and day five, I added fresh media, and then at day seven, I proceed with a cell count and then a phenotype. 

First Set of Data

So this is the first set of data. So here I have the inky side capture. The experiment was run on 96 well plate in triplicate, so that's why you see here there are three images for every condition; so this is the condition without selenium, this is the condition with low selenium, medium selenium, and high selenium. And this is the positive control - if you can hit next Rachel please - so as a reminder, my main CQAs are increased proliferation and increased viability, and also maintains cytotoxicity. So when we run flow cytometry on this experiment, the data shows that as you can see here, we have the cell count, cells per milliliter so as you can see,

We have the T cells - are able to proliferate in media. One that doesn't have selenium media - media  two and media three, the one that has respectively low and medium selenium, but then when we increase the level of selenium to 1.7 milligram per liter, the cells are not proliferating that much and we can see this here on the increasing capture, so using NB-AIR media allowed me actually to improve cell proliferation and cell viability, and then when I checked also for the phenotype to identify the expression of CCR7, if you can click next, you can see here that compared to the control media, the cells expression of CCR7 is higher which translates to less exhaust exertion in these cells. So actually, using this media allowed me to have a quick access to a media that improves proliferation, improves viability, and maintain phenotype and cytotoxicity.

So, using NB-AIR allowed me to create a custom media without selenium which wouldn't be possible unless I would go and buy all the product myself and make all my media, like for I don't know how many components - something around 60 component and it would definitely take me more than two weeks, so using NB-AIR help me reach my goal which is to increase proliferation rate and increase viability and it also gave me this piece of information that it's really valuable because it allowed me to identify the level in me for which selenium is toxic or non-toxic.

So now I know that if I don't add selenium or if I use selenium up to the level of 0.17 milligram per liter, I know that this is not toxic for my cells and then the level if I reach the level of 1.7 milligram per liter, the selenium becomes toxic and this allows me also to develop a small scale protocol because I was able to place an order for only two liters and it also still gave me the possibility to translate to higher volumes in case I want to scale up and work on bigger scale. So, for next steps, I will be testing the impact of other inorganic salts so I don't know if you remember when I was starting the experiment under inorganic salts there was sodium selenite but there was also lithium chloride. So for next step, I might test the impact of lithium chloride on cell activation and cell proliferation which will allow me actually to identify which are the critical component for each CQA and then help me optimize my protocol.

Thank you, that's awesome. Thank you Asma.

We're moving into Saba’s portion of the Media Selection presentation now.

Saba Recap

So now I’m going to tell you about the media that I chose and its properties basically that I checked. Please, so as you know, I use T cells and that's the media that you, that's a cell that you know I usually work with and I wanted to increase the cytotoxicity and basically function of CAR T cells; and then also, I wanted to maintain the viability and activity of the T cells and there are like several components that I will go through, rationale that why I picked that, and in the next slide just to recap from the last webinar.


I mentioned that you know there are multiple factors that impact the efficacy of CAR T cells. You know it could be CAR design or the factors in tumor micro environment, but one of the factors that have full control on it is the quality of CAR T cells and obviously this is influenced by the fuel that we provide for this CAR T cells so, next slide please.

Experimental Outline

Here is my experimental outline. I use two different processes one is the standard process that we use in the clinical sector and in this process we get the T cells and we activate them with dynabeads and after a day we add a lentivirus vector and then we expand them for several days and at the end we can check their functional properties. And the second process is my personal approach that I developed at UPENN and it's the use of non-activated CAR T cells, so in this way we actually do not activate CAR T cells, we keep the T-cells quiescent, we just get the T cells and then we add lentivirus vector and then after a couple of days, we can check their gene transduction and then check their functional properties. Next slide please. So these are basically the components that I picked. I picked IL7 and IL15 cytokine because they are cytokines that permissive for gene transfer for non-activated T cells. When we keep the quiescent cells and we want to have gene transaction, we need to have the cytokine also.

Next I wanted to see the impact of lowering the sugar level in the media because several studies show that the sugar level is not optimal, so that's like another thing that I wanted to see and see the impact of it in the in the CAR T cell function. Also as I explained before, I wanted to check Physiologix because we also showed that that impacts CAR T cells function in mouse model of neuroblastoma. Another component that you know we're very interested in right now is arginine. Again, like you know, they're like studies that they showed that T cells that are grown in media including arginine, has showed a higher anti-tumor function in mass model of melanoma, so that's the another component that I was interested in to see if I add that to my media, can I change any impact of CAR T cell function. And also the last component that I was interested in was carnosine.

Carnosine is actually is in, Physiologix contain carnosine and that's like that control the pH of the media, which is important for gene transduction and cartesian function. Next slide please. So, one of the important determinant of the approach that we use in the clinical sector is the proliferation of the T cells, so in series of like clinical, pre-clinical assay we always interested to look at the proliferation of cortisone and their viability so we do this rigorously at a regular interval with flow cytometry and we monitor also the cell size which is an important determinant of the activity of the T cells. So as you see in the graph on the left which we showed the population doubling that over days like the my control media which is red, you know at by day 10, it plateaued but cells that are grown in the customized media that I picked, they're still growing and this shows that basically that they have higher function.

Also, we can see the cell size which we see that by the end, they maintain the cell size is higher at the end like by day 10. The cells size is higher in the constant that cells are that are expanding in customized media compared to the control media which suggests that the activity is high, the activity of these cells are higher. Next slide please.

Another parameter that I wanted to look is the transduction efficiency of lentivirus using like this media into my two different processes either like you know cells that we activate or you know the non-activated T cells. So on the top part you know we see it in the activated T cells, we see and we use a fluorescent protein MRFP and then we see a similar MRFP expression level in in the activated cartridge of context, you know both of them are like almost the same similar level. On the bottom graph, in the non-activated CAR T cells, no CAR Ts, oh sorry these are like only T cells and then they're the letter viruses MRFP and we actually noticed that you know the transfection efficiency in the customized media is lower than the control media. So now this is important and this is great result for me because it's important to notice that the factors that you know I picked, they're not optimized for this process. This specific process so I have to explore more factors to see how can I boost the gene transduction in a non-activated T cells.

Next slide please. Another important function that I wanted to check was the metabolic activity of the these cells because there is the very high interest in the field about the metabolism and we know that the metabolism is linked to differentiation and as I explained in the previous webinar, that we always wanted to have less differentiated T cells.

That's why actually the second process of non-activated came in because we wanted to just like eliminate the activation which and trigger robust differentiation so, next slide please, so here in the graph we checked the oxidative state of cells that are expanded in the control media or in the custom media and as you see the oxygen consumption rate of cells that are expanded in controlled media is almost half of the custom media,

so it tells us that the custom media actually increased the oxygen consumption rate of the cells which is a great function and it actually tells us that they would in rigorous, let's say environment, they would act a lot better and they have like higher functional ability so now knowing this you know I would really like to explore other candidates that I can manipulate uh for example uh to in the media to see like you know if I can optimize my lentivirus gene delivery and maintain my functional competence so one of the things that you know I was um thinking that you know maybe for that 24 hours that I do use it for the gene delivery we can use the control media and then you know after that we can switch it to the custom media so that in this case you know we can increase we can have a higher gene transduction and also maintain the the functional competence of the cells that are grown in the customized media. And overall, the whole process was very seamless and very easy to apply for future studies so that's also my plan to have different components and see if I can optimize my media further 


Alright, I think we're moving over to Lori now.  Great to see those really interesting results. It's awesome to see how the small changes can really affect the outcome, how that spurs ideas.

Oh Lori, I think you're on mute. 

Always sorry, you said exactly what I was going to start off with and those are really interesting results and I want to thank you guys for having me participate in this webinar because I've learned a ton. For those of you guys that know me out there know that I have a more of a process focus than an assay focus, so I really enjoyed Saba’s and Asma's data, however Cognate is a CMO, so most of our processes that we're contracted to perform are somebody else's. So I selected an internal project for sharing with you guys. Part of the process group within our technology development um job is to provide material for the assays group, so that's kind of where I went. Cognate is a part of the Charles River Company, so we actually can produce cells for a lot of different assay groups when they need cells for development or fit for purpose evaluation.

Specifically for this, we chose to grow some T-regs, which were going to be used for flow assay, so proliferation of viable t-cells with the correct surface markers is going to be, was our primary objective and the next slide summarizes the experiment so basically, what we did was we took a T-reg profile protocol from Miltenyi and Fisher and kind of combined the two and so the experimental design was just using those kits for the T-regs to do the expansion, and specifically I was looking to increase proliferation.

So I'd have enough cells to test while maintaining as I said the viability and the CD4 CD25 and the fox p3 phenotype the bar is a bit low since I was using that type of an assay. If I were to use a functional assay, I would have been way more concerned. I would have had some other more interesting CQAs to put in here, so I did my experiment. As expected, results in some pretty um CD4 CD25 fox p3 cells and the next slide shows that the viability was decent and the morphology was what I wanted not shockingly the cell proliferation was still a little bit better in the proprietary media so proliferation was like I said, a low bar that was just. And so my next steps I think are really exciting because that is to look at the media's to use different things.

Now I get that trying to make the cells do different things so that they work different in medias isn't really everybody's gig and that is it's not even my gig all the time but the ability to tweak the media to engineer the products that will demonstrate varying functionality is such a powerful tool to keep in your you know bag of development tools. It just demonstrates how powerful this can be and that anybody can do it. Like I said, this isn't what we normally do but it was fairly easy and it was fairly quickly and so my next steps are going to be first of all to restimulate the ones that I have and put them in for an MLR comparison to test them a little bit to see if there was any difference in in the functionality but also then to use this NB process to produce other cells for development. Both the process aspects of it as well as continuing to make cells for different types of assays. 

Alright, thank you so much Lori. And that's science right. You know sometimes we get the results we anticipate and you know it spurs a lot of ideas and really allows us to tweak things to try to get that desired outcome as you were stating and you know the perfect example there knowing and that transparency of knowing what's in those custom media is also quite an important aspect of every study and the ability to be able to tweak that to get your desired CQA. So thank you all for working with us and and doing this little online experiment with us. It's been a lot of fun, I hope you guys had a lot of fun. We look forward to working with you guys more going forward. 

Quick Recap

Rachel, just a quick recap, not really a recap but just a list of the other learning resources we have out there that have introduced NB-Lux, NB-AIR to you guys, our customers ,and the audience out there. You'll find the link at the bottom which will take you to those resources and you can watch them, view them at any time or if you have any questions, also have emails at the end of this. Please before you go, reach out to me directly or our [email protected]. If you can advance to the next slide Rachel, just want to leave you guys with a few final thoughts about Nucleus and what we stand for and why, again, we're so excited about custom media. 

Why choose us? We believe in customization, we've been able to improve ordering efficiency expedite times. We believe in the customization and reducing waste and only ordering what you need. Reducing labor and time, individual ownership is huge. Knowing what's in that media as each one of our scientists pointed out is really really beneficial in being able to tweak that media in order to achieve your desired outcome. Consistency, streamlined QC validation, everything goes into the media here essentially you're ready to use it once you uncap it. Scalability - we are there with you from like I said earlier from the bench to the bioreactor - from two liters two to two thousand. Transparency, no hidden ingredients in our products whatsoever - it belongs to you. They're your choices.

We're certainly there to help you if you need it. But certainly your choice as to what goes in that media. And cost effectiveness, right so certainly the smaller batches come with a bit of a price premium but we're there to scale with you and at scale we can certainly help you realize cost efficiency and save you even millions of dollars as you proceed down that pathway. And finally, AI or technology driven - you know we're using cutting-edge technologies, cutting-edge proprietary algorithms in order to really drive and expedite the formulation of your media and we're trying to do this in a very automated fashion, and we look forward to working with all of you out there to even make it better as we progress. You know through the years, so hopefully you guys will reach out to us and let us know how we can help you achieve your goals and reach your CQAs. So I think at this point we would probably move into the Q&A section. I'll turn it back over to Rachel. I think she's going to moderate that for us and again appreciate everyone for joining please put those questions in the chat box or the Q&A box.

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