Productivity. My name is Rita and I will serve as your moderator today. And as your moderator, it's my role to ensure that we make the most of your time with us. I am here today with Dennis Binder, head of Quality Control for cell culture media in Darmstadt. Dennis holds a mass of science and biochemistry from Heinrich Heine University in Dusseldorf. His his early research was mainly conducted at the research centre Euloge and focus on biotechnological expression systems. After receiving his PhD, Dennis joined Merck in 2017, working in cell culture media R&D on new cell culture, media, materials and formulation technologies. Since 2019, he is head in to quality control for cell culture media and Darmstadt, with key focus on testing cell culture media for release and different upstream chemicals for application applicability in cell culture. Before I turn things over to our presenter, I'd like to cover a few housekeeping items. At the bottom of your screen there are multiple application widgets you can use. There you can also find a reaction button, indicated by the thumbs up emoji, that allows you to give immediate feedback on the presentations, topics, or anything that stands out. All the widgets are resizable and movable, so feel free to move them around to get the most out of your desktop space. You can expand your slide area or maximize it to full screen by clicking on the arrows in the top right corner. If you have any questions during the webinar, you can submit them through the Q&A widget. We will try to answer these during the webinar, but if a more detailed answer is needed or if we run out of time, it will be answered later via e-mail. Please know we do capture all questions. You will also have 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 are watching this webinar on demand, you can still submit poll response responses. 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 headset are turned on and the volume is up so you can hear the presenters and on-demand version of the webinar will be available and after it can be accessed using the same link that was sent to you earlier. So that's it for my side. Now it's my pleasure to turn things over to Dennis Binder. Yes, also one welcome from my side. So today I will talk a little bit about the new cell prime recombinants L3 IGF one which can be used to optimize cell culture productivity. So let's look at what you can expect for today. So first of all, yeah, it's a pleasure to tell you about some benefits which this new offering might have. So first of all, you will hear something about physiological response of lasri in the cells and afterwards you can yeah, here's something about how it is able to enhance cell proliferation, viability and production and. Last but not least, we will tell something how media formulations can be optimized using cell prime LR3. So yeah then give going into more detail, we will talk a little bit about the structure of L3 and what these structure will details are meaning in terms of affinity to different receptors and proteins. And yeah, in the end you will see how supplementing your cell culture media with L3 can speed up growth, yeah, increase viability and. Viable said density and overall also protein production. So let's come to our first poll question. Does your current cell culture media have RLR three in the formulation? Yes. Or no. OK. Paul is now closed. Thank you for these results. It's very helpful for us to know where you stand on this. OK, very interesting. So now let's have a look at the structural details and what this really means to the physiological response inside the cells. So first of all, let's have a look at insulin and the insulin like growth factor. So in the middle of this graph you see the structure of insulin and very closely related to this, you can see on the left panel the insulin like growth factor as a structure. So as you might know, insulin and IGF are most widely used performance enhancing media additives. As you might also gather from these two graphics, IDF is structurally related to insulin as you might easily see. And also one fact is that IGF one is a non glycosylated single chain Poly peptide which is consisting of 70 amino acids and also three yeah internal diesel fight bots. So let's have a look at the last three in comparison to. So as you can see here, there's a lot of structural similarity. In more detail, the functional structure is an exact match for the type one IGF receptor, and you see here that this molecule is very similar. However, it contains an end terminal extension containing certain minor assets and moreover in contrast to the IGF. Molecule we showed in the previous slide, it has an exchange of glutamic acid towards arginine. And yeah, this is also leading to the name of this new molecule. So in principle, it's an IGF molecule with an enternal extension of 13 minor acids. So yeah, let's have a look at what this end terminal extension leads to. So these. Structural addition of this minor acid change has different impacts on binding affinities inside cells. So first of all, as compared to the IGF molecule, the L3 has a lower affinity to the IGF binding proteins. This is, yeah, not preferred since those binding effects lead to the fact that the remaining. L3 is reduced, so it's removed from the cell culture media formulations and also the availability of the lasri is reduced. So yeah, therefore this low affinity to the binding proteins is beneficial. Moreover, especially in contrast to IGF, this illusory has an increased, so a high binding affinity to IGF 1 receptors and this. In detail, is the preferred reaction you want to trigger in your cell line and is the end leads to an enabled and enhanced cell growth and yeah, triggers anabolic metabolism inside the cells, so a beneficial reaction. Moreover, L3 has low binding affinity to insulin receptors whereas insulin itself. So it's also part of lots of different media formulations. Has low affinity to the IGF one receptors, whereas it has high affinity to insulin receptors. So yeah, by means of these internal minor acid extensions, the binding affinities are really changed. And in addition to this, we can also say which is also depending on the cell line a little bit there's the Elstree has a lower concentration which is used. In cell culture and typically this concentration is 100 to 1000 fold lower than typical insulin concentrations. And yeah with both molecules with L3 and the insulin you can trigger beneficial reactions inside your cells and you can really enhance the cell growth, the glucose metabolism and also in the end the protein. Production and this is something that we will show you later on by means of different examples. And we also want to make you aware of the fact that the right concentration of the insulin and the last three inside your cell culture is very cell line specific and this is something we will touch base on later. So yeah. We also said that inside the cells there might be different genetic makeups. So for instance what you can see here on the left side you might have a cell which has lots of IGF 1 receptors and lower portion of insulin receptors. And therefore it might be beneficial that you supplement your cell culture with with lots of illusory and. Yeah, less concentration of insulin whereas other cell lines for instance, something you can see on the right side in cell three. So here for instance, you might have a setup that your cell line has a lower portion of IGF one receptors whereas it has a high portion of insulin receptors. And therefore, yeah, the concentration range might be different and. You might use different concentrations in order to increase your cell performance. In the end both both bindings and both. Connections are beneficial and in most cases lead to an improved sub proliferation and also an enhanced productivity of your cells. So later on we will also shortly touch base on an approach how you can for instance find out what your cell line might be might be using and how you can really at best benefit from these two molecules. So let's come to more general benefits of the use of L3 in your cell line. So this is something which most likely will be true for all the cell lines and for all the applications that you are seeing. So in principle in the biopharmaceutical industry it is beneficial to use non animal origin and also serum free components. So as you might know. You have yeah sustainability benefits which are in place. You might have lower lot to lot very variability when using those components. And also yeah there are lots of supply and also ethical considerations which are beneficial to not use serum. So these are the benefits. However in your cell culture it might. Often be hard to maintain or improve productivity in serum or animal free formulations and this, yeah might be a big challenge especially for some really demanding sellers. And with the three, it might be less hard because we do see for lots of different application in increased proliferation of the cells, higher titers, improved cell growth, higher stability as compared to other components and also. Prolonged cell activity. So yeah, when remembering the previous slides on the new binding affinities and what they are triggering inside the cells, you can easily see that lots of applications might have benefits when using the. OK, so enough with these theory. Let's look at real data. So in order to show you real data, we have for this new offering installed to sell essays which are supporting this new offering and also showing that this application might work for different satellites. So first of all, we have installed myoblast essay and the yeah, the key. Of of this method was to really ensure that this material has high comparability to former offerings. Moreover, by using these red myoblast cells, we are using a cell line which is essentially requiring along our street and those are the L6 red myoblast cells. So with this essay setup, we are using DMF 12 high, glucose medium and we are. Starting without any LR3 edition. So yeah, we also see here that most of the salons are quickly adapting to our long history. And um, so therefore, yeah, the assay set up in principle is that we then use medium without serum, so serum free medium with with L3 in this red myoblast essay and it works with different concentration. And the outcome in the end is to really see with which concentration we have 50% of improved cell growth. And you're also some details on the cell. Say so for instance, we are working in an 8 replicates. We are using different concentrations. We are working with this first essay in the 69 well plate, growing the cells for 25 hours. And um, yeah, also then we are switching to to the longest starting with none and then different concentrations of industry are added and in the end the viability and the proliferation of the cells is measured by means of an MTT assay. So Violet coloring reaction which is measuring the metabolic activity of the cells after 48 hours of of stimulation. So, yeah, let's, let's look at, let's look at the results. So as you can see here, we have compared the outcome for different offerings. So first of all, we have performed this L6 red myoblast cell assay with different concentrations with the former offering. We've done this with a new offering with the powder version that is currently offered and. Also with the liquid version and yeah, what we can see here is really highly comparable results to former offerings. And yeah, this is exactly the concentration which is needed for this specific cell line, which essentially requiring the lustri for for growth. And yeah, concentration was found to be as low as 13 nanograms per mill in in average. And yeah, this is something which which shows the comparability and this is done for every lot for every release and showing comparability to other to other alastray offerings. And moreover we have installed quite robust essay by means of entity. So the essay is validated and quite robust and was really feasible for validation. So this is an essay we have in place for every lot release for the, for this, for this new offering. And in addition to that we also are performing another cell assay for every release which is targeting, yeah chose their lines and chose their lines are of broad interest for lots of our customers. So we are targeting many, many applications of of customers and. This is a cell line which is not essentially requiring the alias 3, but is benefiting of the L3 in terms of productivity and in terms of proliferation. So what we did here, we used chosen cell line which is producing fusion protein and for this assay and yeah this is the essay which is really done for all releases of lots is using our cell ventor catalog media. So as a Bayesian medium, we are using for seven to for chow comp and as a feed medium we are using seven to four feet com. And yeah, those were used with with different concentrations of L3 and we could vary the productivity of those cells. However, for the cell assay, we have fixed the concentration and have done a classical fat batch experiment with these new offerings and this is something that I want to show you in more detail in the next slide. So of course, again, we wanted to show that this new offering is highly comparable to the former offering and this is something that you can see here. So for instance, what you can see here in the graph on the left side is the fusion protein production over time. So for this fat batch essay, we looked at day 11-12 and 13. So it's a fusion protein. That we are producing here and as you can see here the values for the fusion protein production are highly comparable between the different offerings which are available on the market. And on the right side of this slide you can also see the viable cell density here in in bars. So in Violet it's again the former offering and in turquoise and. The new offering to sell prime. And as you can see here also those are results are highly comparable and also throughout the cultivation you can really see a very high viability of the cells. So with this application we wanted to target numerous applications of our customers which are heavily using Chotzen culture media and chose cell lines in order to yeah to perform their biopharmaceutical. Protein production and we have here again validated cell assay in place which is done for every lot release in order to show applicability, comparability to other lots and also show you that there is no lot to lot variability in place. We've also done this with another catalogue media, which is, yeah, for instance, the Excel advanced feed. And this again was supplemented with long history. And as you can see here, the last three, yeah is increasing the viability over time. So you see here for the red curve, the last three is missing and again, yeah, for the different. The colors you see, all three was added and viability is higher. Likewise for the viable cell density. You can see here that yeah, the viable cell density is increased when using last three. And yeah, most strikingly you can see here the productivity and again here for the red curve you see is missing. Whereas for all the other colors Lasri was added and yeah. Significantly increased the productivity. And yeah, again this is quite Celine specific. However another example showed in a classical fat batch design using our catalog media that. Tighter production can be significantly increased when using when using laws. OK. Next thing we looked at was the effect of titrating, yeah, the productivity of the cell by means of using different concentrations of of illusory. And yeah, in this setup that we have, yeah, we have here we have again, yeah, catalogue feed formulation, which is silento, 4 feet comp. And you can see here we have no feeding in place for this Violet curve. Here at the bottom we have a feeding but no L3 concentration added to the feed. Here in I think blue and all the other colors you can really see increasing concentrations of the last three molecule and as you can easily see the tighter so the fusion protein production. In this case is increased by increasing the L3 concentration and this is quite easy example which shows what effect this molecule might have to selected cell lines. And also I want to mention that for this experiment we were starting with cells which were not adapted to the alias. So yeah, we see for most of the cell lines a very, very quick adaption to this new molecule and no. No pre cultivation is necessary to see these benefits. OK, so next we tested one of our newest catalogue feed formulations which is savenko modified prime and you can see this here on the graph at the bottom at the top. Sorry for that. And this is a very new formulation which is very easy to hydrate and is also room temperature stable for quite some time and is highly concentrated. And also with this new catalogue feed formulation we were able to show the banner beneficial effect of elastic. So you see here Violet curve with feeding but without addition of additional L3 and all the other curves are with the addition of and again here for the third setup. We we looked at, we again found a boosting effect of the L3 when it comes to the protein production of the cell. And yeah, we could, we could simply show that this molecule is working for all our young catalogue formulations and it's quite likely that it will also function in your specific setup. So let's come to our second question. Our next poll question, what therapy modality are you currently involved in? Mabs, VGT or CGT? Paul is now closed. Thank you again for your responses. They are very helpful for us. OK. Yeah. Thanks a lot for providing your answer. Very interesting. And yeah, last but not least, we want to show you or give you an example how it might be possible for you to optimize media formulations by using. And here are some some quick facts. So of course as I showed you in the beginning, your cell line might be or might have different different density of insulin and IGF receptors on the cell surface. So this is a fact that you have to deal with and there's no generic answer for all the application. Moreover, using a combination of insulin and allostery can elevate the cell performance more than the use of a single component. So it is it might be helpful to combine those two in specific concentrations and you might really be able to optimize your cell culture process. And last but not least, this is a very tough question. How? Yeah, you can really tailor your concentration with using L3 and for instance, also insulin for your individual cell line. And therefore, we do recommend a design of experiments to our customers to really find out what is the best option and what is the best concentration. So yeah, let's have a look. That's how this might might look like. So of course most likely you know that the design of experiments approach is a very powerful tool that can be used to optimize multi component systems in culture, media and cell culture applications. So this setup might be very easy to find the most economic concentration. And what we would suggest here that you fix all other cultivation conditions and use L3 and insulin concentrations in the given ranges. So for instance, we do suggest to start with difference in concentration of factor 200. So as you heard in the beginning, we do expect that as compared to. Insulin, we do expect that lasri concentrations should be 100 to 1000 fold lower as compared to insulin. And in our specific setup here we took as a first guess the concentration difference of 200 fold and we do recommend to go into this experiment with four different concentrations and of course. Since it's cell culture to do these experiments in triplicates to really have robust setup, yeah. And this, yeah design of these experiments could look like this. So for different insulin concentrations for different 3 concentrations and they are differing approximately by factor 200 in this, in this example. And yeah, this is something we would recommend to really test. Your cell culture application for improvement by means of using these two components. OK. So yeah, this is the last thing we would recommend and this brings me to a short summary of all the things that we've talked about today. So first of all, there will be starting from today two new offerings for every purpose in the system. So there will be a liquid offering for this, for this, the article code is shown here and there will be of course also. Older version of this new offering and the article code shown here and what we can really tell you both. Components. Both new offerings are really thoroughly tested, so of course there's a protein assay in place. There are purity measurements, identity measurements. And also we look at appearance and more. Moreover also tradesmens are looked like. So we do report also trace elements on the CFA to further prove that those materials have high suitability or cell culture and cell culture relevant trace elements like iron, manganese or zinc are monitored. Furthermore, we do offer endotoxin bioburden testing and yeah. Most most importantly, we are doing two different cell culture assays to ensure first yeah, high compatibility for child cell lines, which lots of our customers are using. So therefore we do a chow fat page essay as shown in our slides. Moreover, there's a rat myoblast essay in order to really show that there's a really high comparability to other molecules which are there on the market. And last but not least, you will also get this molecule or it will be at least offered that you get self prime dossiers so that you have lots of different information in place to from your qualification of this material and also to cope with with your regulatory proposals. And with this in place, we do ensure that there is no, yeah, lots to lot variability when using this molecule and at best very good performance in your application. So let's sum up. L3 is a non animal origin alternative to IGF market. It can be used in 100 to 1004 lower concentrations and insulin. It has a very good stability since it's not binding to to the IGF binding protein for instance and it is able to to show improved physiological responses. It has proven functionality in lots of different salines. So we talked today about show sales, chosen cells and also red myoblasts. It is offered in two different offerings suitable for different proposals, so liquid and powder, and it's really thoroughly tested by means of to sell essays. For every lot release. And well, that's it from my side. I'm very happy to take your questions and yeah, thanks a lot. Thank you, Dennis, for this great presentation and now it's time to answer questions that have come in from our audience. But before we do, I would like to remind you that it's not too late to send us your questions now using using the Q&A widget. We will try to get through all of them, but if we run out of time, we will respond to you individually. And as a reminder, this webinar will be available on our website soon. All participants will receive an e-mail notification when it's available for viewing. Now back to Dennis. We'll start answering questions that have come in. Do you question what? Do you have any data available using the supplement on cell lines commonly used for viral vaccine production? Mm-hmm. Yeah. So we do have internal data that's common. Cell lines which are used for viral vaccine production such as excels for instance, are very responsive to to the L Street. And I can also tell you that in some internal formulations, allostery is used to support those cell lines and they are in principle very responsive. Of this to the best of our knowledge and for the cell lines we have already looked at. OK. Next question, how much less of? Are LR31 needs compared to IGF one? OK. So in principle this is also something which, yeah, little bit also depends on the cell line and also the receptor and the IGF binding protein density. However, there are studies showing that by means of this end terminal extension, the alias 3 is 2 times more stable in cell culture than IGF. So first guess would be that it's, yeah, half of the concentration for instance, better affinity to the receptors, lower affinity to the IGF binding proteins and therefore approximately half of the concentration would be would be a guess. However, this is very saline specific of course also. OK, let's see. I think those are the only questions we received. Feel free to send us more questions. OK. Thank you very much for all the questions and if we did not get. To you in time, please feel free to e-mail us directly to register for future webinars or to access our archived webinar library, please visit our website and you can also download the presentation slides and the take action field that will pop up on your screen once the webcast has finished. I would like to thank Dennis Binder for today's presentation and thank you to our audience for joining us. Have a great day. Thanks for joining. Bye, bye. _1732459150111

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