Hi, everyone and welcome. My name is Isada Kia and I will serve as your moderator today. Thank you for joining us for today's Webina material attributes of proloxima 188 affecting the application in cell culture and liquid protein formulation. As your moderator, it is my role to ensure that we make the most of your time with us. I'm here today with Mellie Irvin and Alisha Antonella Antonino. Manny Allen is a Strategic Marketing Manager at our company since May 2023. For the past three years, she was having a protein stability laboratory and process solution. In this role, she was mainly responsible for the development of new technologies to improve the stability of protein formulation. Nelly has joined the company in 2020. Her previous work experience includes a post acquisition and analytical R&D adapter. She holds a PhD from Technical University of Dortmund and Chemical Biology. Alicia Antonello is a Senior scientist in the bioprocess, chemistry team and process solutions at the company. She is mainly focused on the development and application of surfactants for bioprocessing, from cell cultivation to harvest and 1st treatments of the product. Alicia turned the company in 2018 from her postdoc. She holds a PhD in Chemistry from Max Planck Institute for Polymer Research and Minds. 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, which you can use. There you can also find a reacting button, indicated by the thumbs up emoji, that allows you to give immediate feedback on the presentation, topics or anything that spans 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. 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Lastly, attendees who wish to receive a Webina certification will need to fulfill the criteria of minimum 30 minutes viewing time and completing 2 poll questions within the duration of this Webina. So that's it from my side. It's my pleasure to turn things over to Nelly and Alicia, and thank you for the kind introduction. Let's start with the Webina and a short introduction of Pollock Samia 188. So this molecule is a tree block copolymia consisting of an hydrophobic polypropylene oxide chain, as you can see here in the middle marked by the blue circle, and this is flanked by two blocks of hydrophilic polyetrylene oxides. According to the Pharmacopoeia, the number of units for ethylene oxide is an average 80 and for propylene oxide it's an average 27. So since this molecule is consisting of hydrophobic and hydrophilic parts, it acts as a non ionic surfactant and it can be applied in different functions. We will focus today on biopharmaceutical formulations and cell culture media. So polyxamia 188 can be applied as a non ionic surfactant in bio therapeutic formulations to stabilize proteins against mechanical and interfacial stresses and in cell culture media. It can be applied in the cell culture of mammalian cells as a sheer stress protector. So according to the Pharmacopeia, poloxamia can have or Poloxamia 188 can have a molecular weight from 7680 to 9510 gram per mole. So within this range, poloxamia 188 is meeting the Pharmacopeia specifications. So if you look at the market now, you can find a variance in the molecular weight of Pollux AMIA 188 for the product from different suppliers but also lot to lot variants for a product from the same supplier. And the reason for this can be the nature of a polymer. So you can have different chain length, different impurities so. Polyxammia 188 is rather a mixture of multitude of structures than this one molecule that you can see on the slide. So now that we know that Polyxammia 188 can have a molecular weight within this broad range, we want to understand. If the molecular weight has an impact on the function in these different applications. So to understand this better we need to have a look at the different stabilization mechanisms of polyxamia. So let's start with proteins, so in general. There are two different mechanisms described how surfactants can stabilize proteins against mechanical and interfacial stresses. So here you can see a protein solution in the absence of a surfactant. So in the absence of a surfactant, proteins tend to absorb to interfaces. So for example if you have a vial with a solution. And you have a air headspace or if you freeze your sample and crystals are formed in solution you have you have ice liquid interface and proteins can absorb to these interfaces and they start to form protein films so they cover the complete surface. And when they are at the surface, proteins can start to expose hydrophobic patches from the core of the protein to the surface of the protein. And these hydrophobic patches can start to interact with each other via hydrophobic interactions. And this can lead to protein aggregation in the presence of surfactants. The. The surface can be covered by the surfactants because they have a higher surface activity. So this means for the first mechanism that is described in literature the competitive surface adsorption that is shown here. In this figure you can see that surfactants cover the surface and the proteins remain in solution and thus. The proteins are prevented to absorb to the surface. Since surfactants have a higher surface activity, they can even replace already absorbed proteins from the surfaces. The other mechanism that is described for surfactants to stabilize proteins is shown here in this figure. So here you can see. That surfactants can also directly interact with proteins in the solution and thus they solubilize the proteins and act rather as a protein chaperoni. So, for example, when proteins expose these hydrophobic patches to the protein surface, the surfactants can start to interact with these exposed hydrophobic patches also in solution. In a recent publication it was described that poloxamia with a higher hydrophobicity can enhance protein stabilization and reduce particle formation in MAP formulations under stress conditions. The explanation for this behavior was that poloxamia with a higher hydrophobicity have a higher surface activity, so this means. The more hydrophobic poloxomia is, the more it's absorbing to the interface and thus it's covering the surface and the protein remains in solution. The other explanation for this was that the higher poloxomia is, the more or the higher the affinity of poloxomia to interact with these hydrophobic patches that are exposed to the protein surface. So now that we know that poloxamia with a higher hydrophobicity is able to stabilize proteins much better, we want to understand if there is maybe a correlation between the molecular weight of poloxamia and the hydrophobicity of poloxamia. But before we answer that question, I will hand over to Alicia. And to show you more about the stabilization mechanism of poloxomia in their culture media, thank you, Nelly. And thank you so for showing us the first application of poloxomer for protein stabilization. And now let's change our our point of view in our application and let's think about psych culture. Usually psych culture can be done for example in spin tubes or in bioreactors. In bioreactors for example, you will have mixing to guarantee in homogeneous medium for the cells and you will have sparking of oxygen to guarantee to provide oxygen to the cells and these will create bubbles. When you don't have any any poloxomer added to the cell culture, you will have the show cells or the cells in general will absorb to the gas bubbles because they have and they are hydrophobic to the hydrophobic surface properties and we know that the bubbles eventually will burst and these so this bubble bursting together with the steering and this parting will contribute to the shear stress. And this is a really dangerous for the cells because since they are quite fragile, they are mammalian cells so they don't have any cell wall. They they will also risk to die and also the bubbles we create the form altogether and they will trap the cells in the form and they will bring them to the surface of the bioreactor where there will be a lack of nutrients and oxygen and one also they will eventually die. Instead if we add the poloxammer 188 to the cell culture, since poloxammer we understood from the first part already illustrated by Nelly, is a surfactant, so it will absorb at the interface between there and the liquid. And since is a big block of polymer for hysteric effect will keep the cells far away from the surface of the bubbles and. And will also, after decreasing the surface tension and also the bubble burst, will be less powerful and this will somehow protect the cells. Furthermore, there's even another theory that also thinks that the poloxomer will interact directly with the cells, so will absorb to the surface of the cells or will insert into the lipid membrane and will decrease their membrane fluidity and this will make the cells much much tougher and that will be even more resistant. To to the shear stress, of course the big question of this webinar is, is also peroximer 188 affected by the molecular weight and Hydrophibicity also for this application. And we can say yes, because we observe in better performing for oximer 188 lots, but performing for these applications. So for cell culture we observe that. In this bed lots there was only in imolecular weight component and also usually in hydrophobic component. We measured the imolecular weight component with a size discussion chromatography and the hydrophobic component with Lcms and we understood the most of the time the presence of a nitrophobic and together imolecular weight component was a root cause of bad shear stress protection and actually we. Kept on making studies on this topic and also we so we observe that an increase of propylene oxide content results in a decrease of the circle to performance. As you can see in this plot on the left hand side you will have on the Y axis the decrease in circle to performance and on the X axis the propylene oxide number. So the number of the blocks and you can see and you can see the keeping constant. The content of you, so the content of the hydrophilic part keeping constant 22 that is also what is common for the poloxone 188 to increasing the molecular weight, we have an increase of the PO content and this brought to correlated with worst cycle to performances. So again we can say that. Even in this case, the molecular weight and adrophobicity are playing a role. That's why we offer three different poloximer 188 in our portfolio and they will target different application. So we have the first on the left hand side that is the poloximer 188 improve expert number 137112 that target the application on liquid formulation. And there's a molecular weight that complies to the pharmacopea and it's measured with iteration that is around 9000, that is between 9100 and 9400 and is the best for protein stabilization. And then we have our two cell culture grade Peroxamar 188 that they are designed for cell culture. So for upstream application and they differ. For the measurement or the measurement method of their molecular weight, in case of the companion, the molecular weight is compliant to the to the pharmacopeia and is measured via titration. And in the case of the seculture optimize instead this measure we say size exclusion chromatography. Both are very good for the seculture protection. And I think after giving you this first overview on the free products that we offer and into main application, I think it's time for the first the pull question. I give this stage to you Isabelle. Thank you very much. And the first call question for you as our audience today is for which application are you using pull out somewhere 188, is it for self culture? For liquid formulation, for solid formulation or for others. And we'll give everyone a few seconds to put in their answers. And I also want to use this opportunity to remind you that participation in poor questions will contribute to fulfilling the criteria for receiving the well enough certification. Thank you very much everyone for participating. Very interesting results. And with that, I will pass it on to Nelly. Thank you, Okay. So let's start with the results. So. As I each have already described, there are different different methods to measure the molecular weight of polyps and so the first one is titration, which is according to the Pharmacopeia, this method can determine the number average molecular mass. Because with this method you measure the number of OH and groups of polyxamia in a sample with SEC SO size exclusion chromatography you measure the molecular mass distribution of a polymer. So this means you get different values for the molecular mass, the number average, the weight average and the peak molecular mass. So for a bimodal distribution, as you can see here in this picture, these three values are different and here MP. So the peak maximum gets gets the most relevant because with this value you exclude all the other species. If you have higher or lower molecular weight species in solution, you will get a different value for the number and the weight average molecular mass, but for the peak maximum it stays the peak maximum and. I'm telling you this because I just want to make aware that you cannot really compare the different values that you can find in the specifications measured by titration and by size exclusion chromatography. You can see here in this results. The size exclusion measurements from our lab because this was the most convenient method for us to determine the molecular weight of poloxomia, and we have used that method for all three products to compare them. And if you compare now these values for the different products poloxomia improve expert in purple. So the one for liquid application and in pink you can find the compendial cell culture. Great and in yellow the cell culture optimized. And if you compare now the values for each product you can see that there is a quite good batch to batch consistency within the product. And if you compare now the different products with each other, you can clearly see that our three different products have a clear difference in the molecular weight and expert for liquid formulation has the highest molecular weight and cell culture optimized the lowest molecular weight and as I told you before in the beginning. And the hydrophobicity plays an important role to stabilize proteins. For this reason, we have established reverse phased HBOC method that was also described in the publication that I have shown you and to determine the level of hydrophobic species since the hydrophobic species are eluting and. At higher retention times they are called here the late eluters, so you can see them here in the chromatograms marked in this blue area. Here above you can see the ample expert for liquid application, which has a quite high amount of hydrophobic species. And below you see the cell culture optimized. Poloxammia which has almost no of these hydrophobic species. So we have also plotted these graphs here and you can see again in purple and proof expert for liquid application, in pink the cell culture great compendium and in yellow the cell culture optimized. And again you see here a high batch to batch consistency for each product. And if you compare now the products with each other, you can clearly see that AMP of expert for liquid application or liquid formulation has the highest amount of hydrophobic species with around 30% and the Sarchartia optimized only 5%, so the lowest amount of hydrophobic species. If we compare this now with the values for the molecular weight from the slide before, we can clearly say that polyxamia with a higher molecular weight contain a higher amount of hydrophobic species. So the explanation in the publication that I described before. For poloxammia with a higher hydrophobicity having a better stabilization effect on protein was that poloxammia with a higher hydrophobicity has a higher surface activity, so that's it means poloxammia with a higher hydrophobicity is covering more the surfaces. So this can be measured by the surface tension. So the more molecules absorb to surfaces, the lower the surface tension gets. So you can see this here in an example that was measured by force tensometer. So in the beginning you can see quite high values. Where the surface tension is measured here as a function of time. So in the beginning only a few molecules absorbed to the surface and with increasing time we can see that more and more surfactants or in this case Pollock somewhere absorbs through the surface and the surface tension gets lower so. If we compare now again the different products here we can clearly see that amprove expert for liquid formulation shows the lowest surface tension values. So this means this product or this molecules have the highest surface activity and for the star culture optimized we could observe the highest values for the surface tension, so a lower surface activity. And what we can say now, if we compare these measurements with the measurements before, is that we could confirm that Pollock somewhere with more hydrophobic species reduce the surface tension to a higher extent. So now we come to the final results for our protein stability and particle formation which was done by forced degradation studies. So we have or to understand better if Poloxomia with a higher hydrophobicity is indeed stabilizing proteins better, we have mixed a model protein with Poloxomia. And then we have stressed these samples by different stress conditions, steering, pump shear, free stall shaking, so different mechanical stresses and interfacial stresses, and then we have investigated the samples after this, after the stresses. If particles and protein aggregates have been formed by using turbidity and flow imaging microscopy measurements. So here you can see an example from our shear stress assay from our steering stress assay where you can see the turbidity measurements. So let me lead you through this graphs here. So you can see in green which is quite low for the turbidity measurements. That's why we have this zoom zoom in here and in green we have the control sample. This sample doesn't contain any surfactants so no paloxamia and it was not stressed so it's the simple map solution. So we have a quite low turbidity value. After stressing the sample which is shown here in Rex, in the absence of Pollux Amia, we have a quite high turbidity value. So this means many many particles and protein aggregates have been formed in this solution. After adding Pollux Amia, we can see again quite low turbidity values because Pollux Amia was able to stabilize. The protein against the steering stress in this test. So for the turbidity measurements we could not not really different shape for our three products but when we looked or had to better look a deeper look into the solution and by measuring the particle concentration by flow imaging microscopy. We could see here a clear trend for the different product. So again we can see here the particle concentration as a function or for the different particle sizes. So starting from 1 to 25 micrometers and again here in green you see the control sample. So no polyxamia, only the map, not stressed. So quite low particle numbers. Unfortunately we could not measure the stressed sample without Pollock Samia, because as you can imagine and or if we look at the turbidity measurements, many many particles have been formed and we could not measure the sample without clogging the flow cell. But you can imagine from the from the turbidity values that there have been many many particles in the solution. Again in the presence of poloxomia the particle numbers after stressing the protein sample was decreased again. And we can see here quite clear that for the purple bars here using amprove expert for liquid formulation. We had the lowest particle concentration formed after the searing stress. So this means if we now look have a look at all our results, we can say that polyxamia with a higher hydrophobicity can stabilize proteins under stress conditions much better than polyxamia with the lower hydrophobicity. And before I have shown you that poloxomia with a higher hydrophobicity has a higher molecular weight. So this means the molecular weight has a clear impact on the function of poloxomia in different liquid applications. And before we conclude, I will hand over again to Alicia to show you the results for the cell culture based essay. Thank you, Nelly. And as Nelly said, we dive in in the airing the results of the Shear Stress protection essay. If also in this case, we wanted to check the performances of Polox number one of the free Polox #188 that we offer in this application and we needed an. A small scale say they could let us to check that without using complicated bioreactor or real scale experiments and it could compromise for that our shake flask, especially with baffles. And the baffles are really important because when we shake the cells in inside the cell culture media in the shake flask, they will reproduce the shear stress. That also can be do. To mix and go starting of oxygen, we use as a cell line the parental choice, but this result has also been reproduced with many other various 2 cell lines and also we use our property. Proprietary chemically defined cell culture media and our minimal requirement that then you will also see later in the plots are the viable cell density is more, should be more than six times to six at the end of the experiment. Viable cells per milliliter and the viability should be more than 80% and so we have this is say what the cells are inside the cell culture media. And these are the results. So you can see in this plot that on the Y axis we have the viable cells, the millimeter and here we have different columns. So this first part it will refer to the BCD and the second part on the right hand side it will refer to the viability. As in the previous slide for the protein stabilization, the purple referred to the improved aspect for liquid formulation, the pink to the companion cell culture optimize and the yellow to the cell culture optimize and we can see that the two. Poloxomer 188, the cell culture optimizer, they achieve, they fulfill the criteria. So they have a very good bi city, more than 6 * 10 to 6 and a very good viability instead we cannot say the same for the poloxomer 188 for liquid formulation. So again we can say that a lower molecular weight but especially a lower hydrophobicity was really the key to. To guarantee the best self protection again after watching this slide of Nelly when she was explaining also the different ways to determine the molecular weight that you can ask why we have two self peroxide 188 the cell culture optimized. We have we launched in 2017 the these approximate 188, the 137097 that is the best in class but this is advise where there are no companions filing restriction instead the companions of culture grade they combine, they provide a good self culture protection but also they are tailored to meet the requirements of the pharmaco. Pharmacophia because the molecular weight is determined by titration. Once more we understand that the the molecular weight and especially even the batch to batch reproducibility and the fact that we provide the three different poloxomer with different level of hydrophobicity and different level of molecular weight. We want to target all the needs of of our customer. You can choose one as a stabilizer for biopharmaceutical liquid formulation and the other two for as a shear stress protect, protect and the force a culture process processes. Since molecular weight and hydrophobicity they have an impact on this application. As a conclusion of this presentation, I will leave again the stage to Isabella for the last full question. Thank you very much, Alita. Coming to the last whole question, we would like to know which one of our Proloxta Mayor 188 products are you currently using for your application? Is it the cell culture optimized, cell culture optimized companion, the improved expert? Are you using a product from other suppliers or aren't you using any Proloxta Mayor 188 products at all? And again, if you are interested in receiving the wedding certification, strongly advising, more encouraging you to participate in those because this will be part of the fulfillment criteria, well again, give everyone a few seconds to put in their answers. Okay, thank you very much also for participating in this whole question. And these are the very interesting results. And with that, I also want to thank Nelly and Alisha for this great presentation. And now it's time to answer a few 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 the 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 after the webinar. As a reminder, this webinar will be available on our website soon and all participants will receive an e-mail notification when it is available for viewing. The slide deck will be available for download at the end of this webinar. Now back to Nelly and Alicia who will start answering questions that have come in. Good. Let's start with the Q&A. And so I see here one question. Could you please comment on the pros and cons of Pollock Samia 188 compared to Preserve 818, So I guess this is referring to liquid formulations for proteins, so yeah. Poloxomia or or let's say police or bait is the main refactor that is used for biopharmaceutical formulations. And however poloxomia is a is a clear and true alternative for police or bait. So we observe quite similar stabilization. Properties for proteins, for fusion, proteins for maps. So for different protein direct derived modalities both surfactants are well tolerated and parental application routes, so both can be applied and so the difference between poloxamia and podisobate. Could be the degradation. So for police obeyed we have a sugar and we have fatty acids that are attached via an Easter function to this sugar and then in compare or compared to or similar to polyxamium we have also pack unity. And if we compare now polyxamia and polyzobate, we see both can degradate under oxidative stress conditions. However, polyzobate contain or has an Easter function attaching the fatty acid to the sugar, and this Easter function can be hydrolyzed. For example by by enzymes, for example from a residual host cell proteins in pharmaceutical formulations. Polyxammia in contrast does not contain any Easters, so it's quite stable against this enzymatic degradation that you observed for the police of it. So here I would say that's a clear advantage of poloxamia in comparison to police of I think I can take the next one that I read here if the Poloxama 188 circular optimize implemented in our cultural media proprietary and indeed it is so is implemented in All in all our cultural media that are that are provided by our company. OK, here's another question. Yeah, what degradance can reside from Pollock Somnia 188? Under which conditions in which have an effect on the stability of? Yeah, I already tried to explain that Pollock Somnia can degradate under oxidative conditions. So this might be for example from trace methods for from UV light from from buffers like hissidine from temperature can also have an impact on on poloxymatic radiation and pair oxides of course can have an impact on. On poloxymatic gradation, however, we try to yeah overcome the stability issues by adding antioxidants to our product. So in our case it's PhD and to reduce the degradation of poloxamia via oxidative or under oxidative stress conditions. And so yeah, different degradation products can can be formed under oxidative degradation. So for example, aldehydes can be formed other reactive degradation products and all these degradation products can again have a negative impact on protein stability. But for this reason, as I said, we add. Antioxidants to to our product to prevent the degradation of polyxamia. I can go with the next one that is this question where they ask if they can make a 10% solution. Of the paloxammer 188 and if this can be also reconstructed in I guess in water not only phosphate buffer, so it's doable to do a 10% solution and also we tried in phosphate buffer and also in distilled water in medical water, so it's possible and and they I. And now also there's an addition to this question that I would be the impact on the pH and I think there would not be impact, but I think that this should be measured okay. So the next question that I see here is. Is poloxamma used as an excipient in commercial biopharmaceuticals? Yes, indeed. Poloxamia is used already in in several biopharmaceutical formulations, so according to our last. View in the marketed pharmaceutical list, we could see that already 3% of all marketed antibody formulations contain poloxamm 188. So as I said, it's already a true alternative for parties of it. I see that there are a few question about the X cell lines. All these tasks that were performed only for two cell lines, so because our the medium that we were developing was mostly for formal media cells, so we don't have data at the moment for for excel lines, OK, I see a question regarding the stability of polyxammia and if. If the pH has an impact on on the stability of the solution, yeah, so of course the concentration of Pollock somewhere itself. Has an impact on on its stability. So the higher the the concentration and the the less the stability under stress conditions. So this is what I can tell you and we have also tested the pH of of different Pollock solutions and so after. After stressing the samples, we could see that the pH or a lower pH, so this is what we have tested, has also a negative impact on the stability of products and solutions. So here is another question about the mechanism of degradation and of poloxamia. And is there an interaction of the degradance with the proteins? Yes, so this is what we have already addressed. So poloxamia can degradate under oxidative conditions, so the pack moieties can be can be degradated. And the degradants that are formed, for example aldehydes can have a negative impact on the stability of proteins. So the proteins can start to aggregate. I see here the question that they ask how recent are culture optimized and optimized companion? I said culture optimized, so the 13/07/97 was launched in 2017 and the companion I believe was launched in 2019 but just or even 2018, but they are, they're well established. So we can we can say that we have been testing them quite a lot and also we have many application. All around the they are implemented was in our proprietary circles for media. OK, so here is a question, how does the polydispersity of Pollock Samia 188 impact the stability for both applications. So I hope we have answered that question with our webinar because we have tried to shown you for each product. That the consistency within the batches are quite good and we have developed these three products to meet the needs of our customers for these different applications. OK, so then there is another question regarding police obeyed and Pollux Samia. So do you see benefits to use mixed fact and systems? E.g. police obeyed 80 and Pollux Samia 188, so we haven't tested that yet because so to be honest. The application of surfactants is tried to be minimized in in biopharmaceutical applications because the tolerability of surfactants in biopharmaceutical applications is usually depending on the concentration. So you try to keep the concentration as low as possible but as high as needed. And for this reason a mixture of two different surfactants, yeah, I I we haven't tested that yet. It would be interesting to see why there would be a need to mix different surfactants if there is another benefit. But yeah, so we we haven't seen that yet. OK. Do you see any other questions each in the chat? Not, not on my side. Let's check. Yeah, so like I I pop up one just now for psychology. And yeah, 80% is also like what what we keep as our parameter? Also as a parameter that we provide for our. So we also test internally our switch batch if it will be compliant with our requirements and 80% is what we are what we said. Good, perfect. So I don't see any further questions. If we missed something, as Isabelle said, we will come to you later and will contact you after the Verina and you can still ask questions later on. So that's also fine for us. Yeah, a lot of great questions indeed. So thank you very much for all these questions. As Nellie said, if we did not get to your question, please feel free to e-mail our presenters directly, but we'll also be in touch with answers to those in case some couldn't be answered just now. Just for closing remarks, to register for future Webinas or to access our archive Webina library, please visit our website. You can also download the presentation slides and the take action field that will pop up on your screen once the web cards have finished. And I would like to thank Nanny and Elise for today's presentation and for taking all of those questions and answering them. And thank you to our audience for joining us and have a great rest of your day. Thank you. Thank you. Thank you. Goodbye. Bye. _1732521521915

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