Hi everyone and welcome. My name is Isabek here and I will serve as your moderator today. Thank you for joining us for today's Webina, how to prevent protein aggregation through Stabilizers and surfactants. As your moderator, it is my role to ensure that we make the most of your time with us. I'm here today with Michelle Seller and to another one Doctor. Michelle Zeller is a senior scientist focusing on novel modality formulation and a life science business in Darmstadt, Germany. She has six years of experience in the field of parental formulations of proteins and novel modalities. Michelle holds a Master of Science and Biomolecular Engineering from T Udamstadt. She then completed a joint PhD program between our company Heidelbeck University and the Steinbes Transfer Center of Biopharmacy and Analysis, during which you work on the characterization of novels affectance for the formulation of therapeutic proteins. Dr. Chan Araman is a Senior manager having the protein formulation laboratory at our Process Solutions department in Darmstadt, Germany. His team is working on delivering solutions to challenges and protein formulations stemming mainly from viscosity and aggregation of proteins. Chan holds a PhD degree from the Institute of Biological Chemistry in Vienna and can profit from a long lasting experience in protein biochemistry, including antibody and ADC development of preclinical studies and clinical trials. Before I turn things over to our presenters, I'd like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets you can use. There you can also find our reaction button, indicated by the thumbs up emoji that allows you to give immediate feedback on the presentation 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 to the QA widget. We will try to answer these during the webinar, but if a more detailed answer is needed or everyone 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. If you are watching this webinar 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 is 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 after and can be accessed using the same link that was sent to you earlier. Lastly, attendees who wish to receive a webinar certification will need to fulfill the criteria of minimum 30 minutes viewing time and completing 2 poll questions within the duration of this webinar. So that's it from my side. It's my pleasure to turn things over to Michelle and Chan. Thank you so much for the introduction and welcome to all of you. Also from my side. Thank you so much for joining today's Swabina. So today we are going to talk about an overview of protein aggregation in general, the underlying mechanism of aggregation prevention by stabilizers and surfactants followed by a deep dive into some exemplary stabilizers and surfactants that we offer within our portfolio. But actually before we get started, we have a poll question for you. So I'm I'm back to give you the first poll question And what we would like to know from you to get started is how often do you encounter stability issues arising from other stress factors rather than mechanical and terminal stress. And that's the kind of a frequency range right here. And you can select from the options between very often, often moderate, occasionally and never. And we'll just give everyone a few seconds to put in their answers. As I said, this will be the first opportunity for you if you want to receive the webinar certification to complete one out of two poll questions. Okay. Thank you everyone for participating these other results. And with that, I hand that back over to Michelle for kicking us off with the presentation. Perfect. Thank you Okay. And with that, we can get actually started with the protein aggregation. So in solution, proteins exist in a dynamic equilibrium between folded and partially unfolded confirmations, and the agglomeration of either one of those species can result in the formation of reversible or irreversible oligomers, eventually leading to larger protein aggregates that can for instance be classified classified as sub visible or visible particles. Thereby, the protein particles can still greatly differ in size, structure, reversibility and solubility, which makes their analytical characterization, for instance, very challenging. Additionally, there are some severe risks that come along with protein aggregation, including decreased therapeutic efficacy or safety issues for patients due to their potential to cause immune responses. Therefore, it is important to take measures ensuring optimal protein stabilization in solution. What makes this additionally challenging, however, is that factors that affect protein stability and that can promote protein aggregation occur during all steps of drug product development and handling. And these factors are also highly diverse and can include, for instance, temperature shift, extreme pH shifts, increase protein concentration, or mechanical stresses like shear stress or stirring stress during the production. One way to achieve protein stabilization towards the variety of different stress factors is the application of stabilizers and surfactants, and their application can be differentiated depending on the stress that actually triggers the protein aggregation. Sugars, amino acids or polios for example, can protect the protein from stresses including thermal stress or pH shifts. There are two mechanisms on how that could be achieved. For instance, excipients can be preferentially excluded from the protein surface, which will shift the former mentioned dynamic equilibrium towards the native state which we want. Or excipients can preferentially bind the unfolded state, preventing further degradation or aggregation. The prevention of surface or agitation induced protein aggregation, on the other hand, can be achieved using surfactants. In this case protein aggregation is triggered by protein absorption to hydrophobic interfaces like liquid air or liquid ice interfaces and thereby a highly concentrated protein film is formed. If this protein film ruptures due to, for instance, mechanical impulses, this will result enlarge protein aggregates in the solution. The protein aggregation preventing capabilities of surfactants mainly come from their surface act of nature, so they will compete with the protein for the absorption at these interfaces and thereby prevent the formation of these protein films. And additionally, surfactants can also be involved in fostering solubility of proteins by more direct interactions. Our portfolio covers both stabilizers like sugars, polios or amino acids as well as surfactants. And in the following minutes, we will deep dive into some of the highlighted sugars and highlighted surfactants to give you an overview of their ability to stabilize therapeutic proteins in liquid formulation and thereby we will start off with the mechanical stress. So surfactants and cyclodextrin, actually surfactants are important stabilizers for biopharmaceuticals. Thereby police survey 20 and 80 are the most commonly used surfactants. Reasons for this fact is their excellent protein stabilizing capability as well as their well known safety profile which makes them applicable in parental formulations. However, polysorbates do have a disadvantage which comes along with their chemical structure and also manufacturing process which is oxidation which can be induced by impurities for instance in the raw substance and hydrolysis and both of those degradation pathways leading to degradation products that in the end can negatively impact protein stability. That is why within our portfolio you can find polysorbates but in a high purity grade on the one hand but also some alternatives. So also among these classical surfactants we offer paloxam at 188. In general, one can say that the surfactant molecules are typically employed at a concentration between .001 and .1% and they mainly stabilize, as mentioned, the protein via the competition for the hydrophobic interfaces. In addition, what you can also find in our portfolio is cyclodextrin, which has been proposed as an alternative to classical surfactants in general And this olicosaccharide has been shown to be capable of preventing surface induced aggregation at a concentration of already 2.5 milli molar. In order to test the protein stabilizing capability of surfactants and cyclodextrin, we perform various force degradation experiments and thereby the initial goal is to trigger protein aggregation in samples that are not stabilized as shown here on the right. So our model protein map E shows increased turbidity and decreased Vonamac content after being stressed by stirring, shaking, pumping of three star cycles and this indicates protein aggregation and degradation. Using this approach it allows us to compare the results of the samples without additive to the results of formulations containing our surfactants or cyclodextrin. And what we can see here on this slide is that our high purity polysorbates 20 and 80 as well as our our alternatives poloxamide 188 and even cyclodextrin managed to prevent protein aggregation upon all stress types that we applied triggering surface or agitation induced protein aggregation and you can see some exemplary results here. So the successful stabilization of MAP E by all stabilizing molecules is indicated by showing 100% monomer recovery and a low turbidity. One additional message to point out here, cyclodextrin, which again does not belong to the category of surfactants should be applied in a different range of concentration just specifically optimized for this type of excipient. To sum up this section in our portfolio, a quick overview. You can find Poly survey 20 and 80 in a high purity quality to minimize impurity induced Poly survey degradation. And even though they show excellent protein stabilization properties and are well tolerated in parental formulations, that structure related characteristic of being prone towards oxidation and hydrolysis have led to the evaluation of alternatives. Therefore, in our portfolio you can also find Paloxima 188 and cyclodextrin as suitable alternatives in liquid formulations of biopharmaceuticals. However, we do have more. So you are now getting a sneak peek into our new product development which for now we will refer to as surfactant 1. So as shown here in this first slide, surfactant one is capable of stabilizing the model protein map E as good as or even slightly better than the benchmarks, which is 1 crucial factor when we are considering and considering an alternative surfactant. Most importantly, however, the goal of this product development was to identify a new surfactant with a higher solution stability than polysorbates and in order to test its stability again, forced degradation experiments were performed. But this time the goal was to trigger degradation in polysorbates, enabling the comparison to the new surfactants performance. So the surfactants were thereby stressed thermally by the application of two enzymes inducing enzymatic hydrolysis or the surfactants were exposed to UV light or hydrogen peroxide to trigger oxidation. And the subsequent analysis of polysorbate or even paloxima 188 indicated here on this slide shows the degradation upon these oxidative conditions using one milli molar of hydrogen peroxide surfactant. One on the other hand remains stable towards all applied stress conditions and that we tested. So currently we are offering the opportunity of an ALPHA trial. So feel free to contact me, my colleague Chan or Superiati have us For more information. And before I hand over to Chan for the next part, we have a second poll. Question for you. Thank you, Michelle. And the second whole question we have for you is what are your primary concerns when it comes to using novel exceptions in your bio therapeutic products? And you can actually select multiple answers here. So that's a multiple choice question. Is it safety and toxicity, efficacy and stability, regulatory compliance, cost and availability or others? And if it's others, you can use the Q&A window that you should be seeing to specify. And again, we'll just give everyone a couple of seconds to put in their answers. Also, again, taking the opportunity to remind you that if you're looking for that Webina certificate, kindly participate in the poll questions. Okay, Thank you everyone for your answers. Very interesting results. And with that, I hand it over to Chan now. Thank you, Isabelle. Thank you, Michelle. Yeah. So also from my side, welcome everybody to our webinar on protein aggregation and on stabilization of protein aggregation. And I will talk about the thermal stress and how to prevent aggregation through thermal stress. So as Michelle highlighted earlier, during this webinar amino acids, sugars and polyols are part of our stabilization portfolio. As such, these excipients contribute heavily to prevention of protein aggregation stemming from thermal stress. Yeah, So let us start with an example from amino acid field. So glycine is one of these amino acids which can be used for parental applications. It is, it can be used as a protein stabilizer and also as a buffering agent. It's stabilizing properties stem highly from its preferential hydration behavior. And what we have here is that to decrease manufacturing challenges, we recommend to use granulated glycine, which shows greatly reduced caking, better flow availability and processability as well as good dissolution behavior and stability during transport plus storage when compared to the crystalline glycine. So for that reason we would recommend to use this product for stabilization as well As for offering reasons. But as I said in the beginning or as we highlighted quite often is that not only amino acids are playing a role in the stabilization portfolio, but also sugars And two most commonly used stabilizing agents among sugars to benchmark excipients are sucrose and Trelos which are also included in our portfolio. Let me start with sucrose. That's a non reducing sugar and that does disaccharide which consists of glucose and fructose which are glucosythically bound and it has very good stabilizing properties, especially in liotilization which is quite similar to Trellos which is the industrial benchmark on this case. In terms of the liotilization, sucrose, which is a stabilizer is often combined with a bulking agent such as monitor or even solve it at all. It is widely used. It is present and 246 marketed and approved injectable formulations of which 239 biomolecules and 333 are small molecules and it's also present in the most biomolecule formulations as you might expect, since it plays quite a big role in the livalization process, 59% are livalized formulated products and 16% are liquid formulations. So it is a must have product in the stabilizer toolbox for each formulation scientist. Nevertheless there are certain risks and there has to be a risk assessment done. So the raw materials contribute to general bio burden and endotoxin risk and there is a potential negative impact due to non particulate impurities on its analytics and stability of the biomolecular formulation. In the end, in the final formulation for those who are maybe not familiar with the nanoparticulate impurities, these are agglomerates of different kind of impurities, proteinaceous or nanoparticle impurities, which can affect really the analytical grade and the stability of proteins in solution as well as in the final formulation. So our solution to that is actually having risk mitigation and manufacturing of sucrose so that we can enhance the quality of the product. So we would like to reduce the bio burden and end the toxin levels and also we would like to reduce the Mpis in other particulate impurities. For this we developed the manufacturing process in a controlled production environment for high risk applications. So sucrose which is derived from beetroot can be dissolved, can be filtrated via technical flow, filtration can be crystallized. And request wise after centrifugation and drying steps as well as packaging steps, we will have the improve expert product which is which highlights actually total aerobic microbial count the tamc and the total east and Mal count the YMC which is assessed by the US for Mokopia And this will be 100 CFU per gram for tamc and lower than 10 cafu per gram for the YMC. And also the endotoxin levels are in alignment with the USR Mokopia values in that so much similar to sucrose. Trejolos as I said, is a benchmark in the liophilization field. It is a disaccharide as well consisting of two glucose which are again glucosidic bond and it is a protein stabilizer. It has very good stabilizing properties, again analogously to sucrose. And for the liophilization, it is a perfect alternative or a perfect agent for faster levelization cost, efficient freeze drying processes. It is quite often used in small molecule formulations in Optel mix, eye drops, hydration. For the tear film it's quite good in liposomal formulations where it as a stabilizer during levelization. So again we have a master product in the stabilizer toolbox for formulation scientists. Nevertheless, here we have some risks as well quite compatible to the ones which we have with sucrose. But most importantly we have one manufacturer which dominates the global trail lows market. So this is one of the drawbacks what we have. Trail lows is present in formulations at high concentration, so it is highly concentrated. That means during the parental administration it is also going to highlight a high risk application due to these factors which I highlighted. So again similar to sucrose, the risk mitigation in the manufacturing process of Trellos, we can get technical grade Trellos. We can use the similar or even analogous state-of-the-art manufacturing process what we have in Darmstadt and can generate and improve expert product in a controlled production environment for high risk applications. In the end from solution again filtration, crystallization, centrifugation and trying steps as well as the packaging steps. And as you can see the resulting specification meets again the expectations and the assessments, the limits of the US Pharmacopoeia. So let's keep up with the sugars. And so let us show you an excipient, an established excipient which can be used for a new application. So myoinositole is a cyclic sugar alcohol which is present in the human body, especially in the brain and it derives from the glucose as you can see it also on the structure, So it can be used in parental formulations. It has been shown that it's already an excipient in two FDA approved parental drug formulations. There are also other applications for MYO NS at all as active ingredients. Nevertheless, these are not FDA approved as of now. So we had the idea of using MYO NS at all in a new application as an alternative stabilizer for protein formulations so that we can prevent aggregation which is induced by term of stress. The idea in general stems also from the idea that sucrose and trialos are used to prevent and reduce aggregation in protein formulations via increasing the melting temperature and therefore stabilize proteins and formulations. So thus that my you know it all might overcome potential stability challenges with new protein trapeutic entities where known excipients such as sucrose and trialos may not perform satisfactorily. So with that in mind, we designed experiments with model proteins such as standard monoclonal antibodies, fusion proteins as well as Adcs. And the design of experiments or the experimental design in general was founded on the terminal force degradation studies. In the first step we had our model protein, we had different excipients for stabilization reasons and we assess the melting temperature as a measure for unfolding of proteins protein stability. The results from these studies helped us to develop the second part of the experiment where we used again our model protein different excipients in a isothermal stress under isothermal stress conditions. Whereas we use the melting temperature which we took from the first step and we were 10 degrees below so that we could have a more general approach for the level of thermal challenge which is comparable across different protein models. And at that point we measured the pH. We looked into the osmodality of our buffering solutions, of our formulation solutions. We looked into different aspects of monomer content or protein purity as well as the content of protein, how much protein we are losing using chromatographic methods as well as occurrence of particles which was assessed by turbidity. To give you the idea how it looks like in the end and how we performed these experiments, here are the results for the thermal transition temperature or the melting temperature as we as we can call it here as well how we determined that we used the nano DSF approach for that. And the transition temperature is the temperature at which the protein starts to get unfolded. So as you can see the reason for having the -10 degrees among different kind of proteins stem from the idea that they were more or less at the same level at these temperature, temperature range in both pH values at which we use that. More interestingly for us the increase was similar to the benchmark substances for the myoinosital as we used it here when it was compared to Circros and trail those at both P H's. So having proven the first hypothesis, we want to look if our second hypothesis could be proven as well in terms of that the that you know the tool is going to perform in different kind of analytical experiments either as good or better than the benchmark excipients for protein stabilization stemming from thermal stress conditions. So just you, I mean it's a quite crowded slide. Let me help you through the slide. As I said, we measured turbidity, we measured the content of a monoclonal antibody. In that case, as you can see it here, using size exclusion grammatography, we went through monomer purity to see how pure the monomer was when we were using our excipients and when we were not using the excipients. That's the control sample which is in which is in purple and is referred to here as buffer and Maya Innocent. Also, CROs and Trejolos are in blue-green and magenta respectively in each diagram, as you can see. So under the stress conditions, it is expected that the turbidity is increasing. Nevertheless, this increase could be kept in a level which was acceptable after 72 hours when we were using stabilizing excipients. The same holds through as well for the loss of monomer content or the monomer content beginning of the experiment as well as the end of the experiment after three days where we performed with stabilizing exhibits better after 72 hours. After three days in terms of the monomer content, it was increased compared to the non excipient containing buffer. More interestingly, the monomer purity was even better in that case when we compare the content and the purity results. So we had quite pure monomers even after three days with which we could work. And the aggregates, the amount of aggregates which we could see was significantly reduced compared to the formulation without the excipients. So we can say in retrospective now that the monoclonal antibody is stabilized to the same extent using my as at all as with both benchmark reagents sucrose and triallos. Just to give you an overview of how it looks like actually with also different types of proteins as I said in the beginning of a couple of slides ago. So we used fusion proteins, we use monoclonal antibodies, we use antibody drug conjugates and we can say that my own as a tall performance is comparable in some cases even better than the benchmark excipients with different types of proteins. Oops, sorry. And with that, we will have another whole question before we go to the end part of the presentation. Thank you, Chan. And our last whole question for you today is how likely are you to try a new or novel excipient in your bio therapeutic product? Is it very likely, somewhat likely, rather neutral, somewhat unlikely or very unlikely? And again, if you're looking to get the webinar certificate, this is the last option to go for the pull questions bit. So we'll give everyone a few seconds again to put in their answers. Okay. Thank you everyone for participating. These are the results. And with that, I'll turn it over back to Chan for the last bit of the presentation. Well, thank you Zabel again. Yes. So as I said, we are at the end of our presentation. I would like to summarize it at least this part and afterwards I would like to give you an overview of our portfolio and summarize the whole presentation. So I hope I was able to give you an overview of distinct protein stabilizing excipients including sugars, amino acids and polyols. Hereby sucrose and Trejolos increase the transition temperature and stabilize proteins and formulations and are used in a large variety of parental applications. These are benchmark substances with which we operate. Alternative stabilizers are also in our portfolio and I was able to show you today Mayo Inositol which is used as an excipient for different reasons for more liability reasons in the past and it shows also comparable stabilization effects to benchmark substances on different monoclonal antibodies, a DC's and fusion protein samples. We have also additional stabilizer excipients which are evergreens such as amino acids as well as bulking agents, tonicity agents for liberalization formulations in our portfolio in improve expert quality and all of them are utilized in parental applications as well. So as I said now the final summary, our executive summary of our presentation. So here you have an overview on distinct type of stabilizers from our portfolio separated by the mechanism of stabilization such as thermal or bulking gauge and tonicity as well as mechanical stress. And on their performance, our diverse stabilizer portfolio helps us to find a tailor made solution for most of the problems encountered during formulation development stage. And I hope that you enjoyed this webinar as much as we did and we will be happy to take any questions which you had in the Q&A session as well as from your side. Thank you. Thanks a lot and thanks for listening. Thank you, Mr. Lin Chan for the great presentation, very insightful. And as John said, now it's time to answer a few questions that have come in from you as our audience. But before we do, I would like to remind you that it's not too late to send us your questions now, so you can still use that Q&A widget. And this also applies to on demand viewers. We will try to get through all of the questions, but if we run out of time, we will respond to you individually afterwards. As a reminder, this Webina will be available on our website soon and all participants will receive an e-mail notification when it is available for viewing. Now back to Michelle and Chen, who will start answering questions that have come in. Yes, I can start with the questions. So thank you so much for the questions. They're actually excellent questions coming in already, and the first one that I saw is what approaches can you incorporate in the downstream purification process to prevent aggregation, like in TFF concentration. So there are several things that you can do, for instance the operation conditions. So use gentle operation conditions like low shear rates, low trans membrane pressure and also try to keep the temperature low to minimize the potential aggregation. Then the membrane selection. So this is crucial. A smaller pore size for instance can already help retain smaller aggregates. And then also very important are actually the buffer conditions. So buffer conditions including the ionic strength, the pH of the buffer. But also I mean perfectly for this topic is excipients that you can already add for instance surfactants, polymers and also cyclodextran for instance. And I can actually go on with the second question because it's kind of follow up question that fits quite well. Do surfactants get concentrated during ultra filtration and dire filtration? Yes, actually they do. That is an issue. They can concentrate, they can change the composition. It really depends on the surfactant it and many different factors like what type of membrane you're using, what type of or the pore size of the membrane that you are using. So this is actually something really to consider when doing that. So perfect and I will take over with another question. We have a question as well on the usability of our surfactants and stabilizers. So the question is, are your novel surfactants and stabilizers in the commercial phase? Is there a publication that can be consulted about these novel surfactants and stabilizers? Do you have the analytical method for quantification? So it's rather a large question as you can see. But so the answer to this is for surfactants as Michelle already highlighted how our portfolio looks like. And there is one surfactant which is not on the commercial phase yet, but which is more on the R&D phase, on the trialing phase and that's the surfactant one. And this presentation, all the other surfactants about which we talked are already in the commercial phase and are being used not only by us, also by others. There are several several publications for novel molecules. So for instance, in case of Myo Inositor which is used as a novel stabilizer, there are publications of how it can be used in liotilization processes or in in different in different applications such as such as drying processes. And nevertheless it's used as a term of stabilizer and how it works. There are the best of my knowledge no publications on that. That is an assumption which we encountered in our labs when we were doing the work when we were hypothesizing it. So in terms of that, the publications which we shared on our slides are accessible. These are publications which gave us the hint to look into that area as well. And the last part of the question is actually for me a very, very nice question because we have analytical methods for the quantification. And as you can see, for instance, we are working with HPSC quite often and we are working also. With different methods of HPLC, it is not only limited to to size exclusion chromatography. Another method will will be for instance reverse phase chromatography where we can look at it or we can use different kind of detectors to to measure to measure molecules such as such as refactants as well as small molecules. So these possibilities are there and we have indeed quantification methods for for our excipients which we presented here, regardless of if they are commercialized or not. Okay, then I can continue with the next question. So I have as Paloxama 188 is stabilized with 70 ppm BHT, why is it prone to degradation? Actually a very good point. So on the slide that I showed where you could see the degradation of Paloxamel 188, it was a very harsh condition, so it was one milli molar of hydrogen peroxide and the samples were also incubated for 21 days at 45 degrees Celsius. So the 70 PPMEHT here, it just wasn't enough to to prevent oxidation entirely. And since Paloxamet 188 does contain the pack, that's just something that is going to happen. However, that is actually the advantage of paloxima 188, especially with the 70 ppm BHT that it's not as as prone to degradation via oxidation as polysobates. And also a good advantage is that here we don't see hydrolysis of the molecule. So that's something that is just structure related and also in our experiments we did not say see any hydrolysis. So clear advantage over the benchmarks police over at 80 and 20. You have another question Chan or there is one question which I would like to answer and this is a question which is which actually gives. So which I really liked it was about sorry for that. Is there any interest in including history in the portfolio in the future? If not, is there any particular reason for that? So basically we have history in our broader portfolio and it is used also as an amino acid. Yes true, it is used for stabilization reasons. Nevertheless it is also acquired common buffering agent. So using it as a buffering agent and this in the same quality as we have it here. It can be used also For these reasons. Of course it is not included in the stabilizer portfolio as such because it is mostly used for as a buffering agent. That's why we didn't include it. But yes, of course Histadine is also part of our portfolio and can be used For these reasons as well. OK, then I have another one regarding surfactant one, does surfactant 1 still have the hydrolysis problem as police orbates? And here I can clearly say no, it doesn't. So at least throughout all our studies, so the enzymatic hydrolysis that we did, but also at an elevated pH that we tested, we did not see any degradation of surfactant one in terms of hydrolysis. So I have one, I see one question about, can anybody hear me? Yeah, I see one question about the success of new and novel excipients in the clinic. So that's a question which can be answered depending on how we, how we define new. So a new excipient such as when we are talking about alternative stabilizers such as myo inositol, right. So this is quite a nice example to give. It is it has been already clinically proved in different trials for as an inactive ingredient as well as as a nutrition supplement. So meaning we can it is FDA listed, it is on the grass, so it is seen as a grass but that doesn't necessarily mean that it can be used in different countries for different reasons. So that means if it's already FDA listed in the inactive list of the FDA, then it can be used as an inactive ingredient in different formulations under different usage. And and this is the case with for instance, this is also the case with other excipients. I mean this was some data which was due to time reasons could not be shared here. But we tested also different excipients for our portfolio, also different excipients which are belonging to these categories which we presented today. And there we had also clinical data on how safe these agents are and basically in our approach we will always like to go for inactive ingredients if we know that they have been already used for different purposes as inactive ingredients and excipients. We will always would like to make sure that there is clinical data on that and the clinical performance is favorable if possible, if it's not possible, if it's on the pure R&D side. We are more looking into different essays with close collaboration of our toxicological department as well as our regulatory department to see what kind of essays are needed so that we can get the information on that and then work with that either together here in our company or we'll look for clinical trials if there are possibilities. A very good question, actually okay, I have another good question, when do we recommend polysorbate and when do we recommend cyclodextrin? So it actually really depends on you if you are looking for a replacement for the polysorbates due to the disadvantages that we just have with these types of molecules and something that is also related to the first two questions that I had during the filtration. For instance, if you are running into issues with your police orbate during TFFUFDF and that is something where you could consider testing the cyclodextrin. I had one question which was quite interesting but maybe just already been. Oh yeah, I thought I saw one question which I would like to answer but the question is not there anymore. Then it could be that it has been already answered but not by you Michelle. This I will have heard so but but the question was referring to the method to see how we are, how we are determining the, how we are determining the melting temperature or the transition temperature. So we are using nano DSF, nano differential scanning fluorometry and we measure the intrinsic fluorescence of Tryptophane. And depending on the change of the intrinsic fluorescence of the Tryptophane, we can then assess the the temperature of aggregation as well as the temperature of unfolding or the point of unfolding. Why are the changes on the melting temperature okay. So thank you very much for all the questions. I think John and Michelle, I took a lot of them. Thank you both also for answering. If we did not get to your question, please feel free to e-mail our presenters directly. And I'm just going to pull up their e-mail addresses so that everyone can see them. In case you're interested in participating in the alpha trials, which Michelle mentioned earlier, or also if you'd like to request product samples to test in your labs, feel free to follow the button along with the respective banner you see on your screen and reach out to the team. And to register for future webinars or to access our archive Webina library, please visit our website. You can also download the presentation slides from today in the tech action field that will pop up on your screen once the webcast has finished. And again, I would like to say a big thank you to Michelle and Chen for the for today's presentation and also for you to you as our audience for joining us and wish you a great day and hope to see you soon. Again, it's not like you. _1732211241295

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