Hi everyone and welcome. My name is Navidrafania and I will serve as your moderator today. Thank you for joining us for today's webinar AAV Process Intensification using High Salt Lysis and Benzonase Salt tolerant Endonuclease as your moderator. It is my role to ensure that we make the most out of your time with us. I'm here today with Sara Lucia and Dimitri Zebazinski. Sara is a global strategic marketing manager specializing in manufacturing enzymes and downstream chemicals joining process solutions in 2017. She today uses her expertise bringing to life new products for a wide range of pharmaceutical segments. Sara Lucia holds a Master's degree specializing in cell and gene therapy from the UPMC and Ecol nachmal superhero to Paris. She also holds a Master's degree in Health Marketing from the Sophon University. I'd like to note that Sara is currently battling a minor sickness which might affect her voice. If during the presentation she finds it too straining, her esteemed colleague Nikolai Stankiewicz will step in to continue on her behalf. We're grateful to Sara for her dedication and pushing through today, and we're equally thankful to Nikolai for being ready to assist. I'd now like to introduce our next speaker for the day, Dmitry Zebezinski. Dimitri completed his PhD in molecular Genetics from the Weismann Institute of Science, followed by a postdoctoral experience in the antibody drug conjugate department of our company. He worked on mode of action discovery, payload ranking, optimization and genetic engineering of cellular models. Later, he transitioned into cell culture media development as a senior scientist, where he focused on raw materials, Cho cell line development and metabolic engineering. Currently, Dmitry is a senior scientist in the Novel Process Materials R&D group, where he specializes in enzyme engineering and process development. 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 our 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 the full screen by clicking on the arrow in the top right corner. If you have any questions during the webinar, you can submit them through the QA 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 call 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 all responses. 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 speaker or headset are turned on and the volume is up so you can hear the presenters properly. An on demand version of the webinar will be available after and can be accessed using the same link that was sent to you earlier. With that out of the way, we now start our webinar with the first poll question. Our question for you is what therapeutic segments are you working on? Vaccines, viral gene therapy or others? If you're choosing this option we'd be happy to see your responses in the Q&A section. You don't have a few seconds to choose your response and or write it for us in the Q&A. OK. Paul is now closed. Thank you for these results. It's very helpful to know where you stand on this indeed. So that's from my side for now. It's my pleasure to think to turn things over to Sara Lucia. Sara, thank you very much for this introduction. So hi everyone, very happy and excited to to be here today. So I will guide you through the introduction and some key points during this presentation before handing over to to my colleague Dmitry for the application part. So before we dig into into the topic where does that webinar falls, it definitely falls into the improved process efficiency buckets. So as a life science provider, we are always trying to find new solutions to make sure the drug manufacturers are getting highest yield up possible. And this is definitely here that we are folding today and this is the message that we would like to to bring. So before we dig into the topic, a first point would be about the checklist, because today we will talk about nucleases, we will talk about celluliser steps, DNA digestion steps. One of the key chemical that you're finding in those steps is nucleases, so an enzyme. So it's always good to keep in mind what are the key criteria for enzyme selection, especially for AAV manufacturing, because we know AAV is always very close to patient bed and there are some very specific regulatory guidance. So the point of this slide is really to say that GMP is not enough. GMP is definitely the baseline right when you're looking for raw materials, but it's definitely not enough. The purity is very important. The glycosidylation status is also very important, right to make sure your enzyme is devoid of post translational modifications. We'll come back to that point. The bio burden aspects are key as well, Mycoplasma adventitious viruses and the toxins. So all those criteria are definitely complementing this GMP claim that is only connected to the manufacturing. So we can also have a target samples to make sure that there is no contamination to your bulk product like the suppliers, logistic, anything connected to supply robustness, Technical Support and of course detection methods. So you see here you have 10 points. And that means that again, when you're looking for raw materials, you were looking at enzymes. GMP is not enough. We need to look far beyond GMP. So today, we will focus on a specific unit operation that we like to call midstream. So that means we are after the cells have been produced, after the vectors are produced and before purification. And that step is actually very critical because it combines 2 things, cell lysis and nucleus treatment. So if we have a bit of deep dive into this midstream unit operation, 2 phenomenon should happen one after the other before you can start your downstream processing. The first one is cell lysis, which means that you're going to lysate your cells to recover your viral vectors. So we are really here talking about AAV production. The main way to achieve Cellulitis is with detergents, right? Detergents have this ability to destroy lipid bilayer, which you're going to find in most of the cells membranes, and this is definitely the first step to release your viral vectors. However, while you release your vectors, you're also releasing a lot of other debris including also whole cell DNA that you don't want to have. And here we connect with the regulatory guidance from the FDA. We're actually DNA is considered as a contaminants and it's something that should be closely monitored in the final drug product. So that's when you enter the DNA digestion problem where you need to get rid of DNA but also make sure that those excess nucleic acids are not going to fool your membranes or your chromatography resins. So then we move to cell ISIS. And here what are the key challenges for cell ISIS? There are a number that needs to be kept in mind. The first one is to find an appropriate detergent. The two main detergents that are used, at least from what we see it's right on X 100 and 2020. Those chemicals that are quite old, the Triton X 100 is banned. So we have a specific that webinar on that topic if you have an interest. But the point is that Triton X100 can no longer be used. So if you've been using Triton you will need to find eternities. Also other detergents like between 20 police obeys 20 can also create some concerns especially for the downstream. So picking the right detergent is definitely one of the key aspects. The second one is that your lysis method, whatever it is, needs to not damage your viral vectors. And here on that little graph, what you're going to see is with some very well known detergents, the impact on the use of those detergents on the viral particle infectivity. And so the point is that you're really looking for detergents that is not going to impact your viral vector infectivity. And the last points when we're talking about Cellulitis and detergents, it's downstream cause the fact that the detergent is efficient for Cellulitis, again it's not enough. You need to be able to remove it easily, to detect it easily. And you need to make sure that the detergent is not having any kind of negative interaction with your downstream processing, which we know can theoretically be the case for 2020. So those are the key challenges for sizes. But now we will start entering the topic, right? What about salt concentration? Is salt also one of the key aspects that should be addressed? And here we have actually some literature that can help you answer those questions. So historically when we're looking at silices for AAV, license was performed at 150 millimolar of salt, which is the physiological concentration for the silices. However, there are a number of papers and a growing amount of papers that are showing that when you increase your salt concentration, there are a number of phenomenons that are happening that are going to reduce the AAV aggregation and therefore increase your yield after purification. So it seems like there is a definitely a direct impact between the salt concentration and the process yield. We will come back to those points. Now if we talk about DNA digestion, what are the key challenges? The first one is what we've discussed at the very beginning, how to source to write raw materials, right, the material that has the right quality claims that have the right supply robustness and and make sure that the raw material that you introduce meets all the characteristics that are needed. The second point is about process optimization and nucleus removal, because here again, once that you introduce an enzyme for DNA digestion, you need to make sure that you can quantify it. You need to make sure that you optimize it to not overuse this raw materials. We can help you for that, right. If there are any questions, there are a number of application notes out there. We have a lot of biotech engineers that can support, but this is also a key challenge And the last one will make the connection with this entire discussion is about necklace activity, nucleus activity. And here I'm talking standard nucleus activity is not very powerful at high salt and this is where we're going to enter. The main issue right, is that nucleus, standard nucleus are not active at high salt conditions. So if we spend a bit of time on it. So this is again your little graph showing a drastic reduction of nucleus activity based on the salt concentration. We need to be careful. We see a number of, I would not call them papers because they are not really scientifically driven, but there are a number of communications at the moment saying that some standard replays could work at high salt. This is not the case. The reason is very easy is that at high ionic strength the interaction between the enzyme and the DNA does not happen, so then the cleavage does not happen. So when you're looking at that, if you're looking at nucleus at high salt, what you need to be very careful about is the length of the final DNA fragments that you are getting after digestion. If we spent one second on the right side on the on the of the slides you see the monograph we've the definition of enzyme activity. This is the monograph of the standard benzonase that emerge. Millipore Sigma has been selling for many years. Benzonase cleaves all kind of DNA. I'm not going to get into all the details. What we're interested in is to only go nucleotides of three to five base pairs. This is where the issue stands. If you have an enzyme that is not able to do that, you cannot consider that an enzyme is active at I salt. So this is where we stand. And so now I'm very excited to say that to overcome actually this challenge we have been working for many years on bringing a new member to a benzonase family. And so this is done now with the benzonase salt tolerance that is going to be launched very, very soon. It's a new enzyme, completely new enzyme. It's not the same as the benzonase that you know and it can digest all kind of nucleic acids up to one molar. So this is definitely here revolution, non animal origin iPad, GMP and Ampere experts. So this is just the intro and I will now hand over to my colleague Dima for the application part. Thank you Sarah for the chat, such a kind introduction about the whole process of AV production and the challenges and where do we stand. So let me guide you through so heavy science data about the enzyme and what it can do. So when we identify the enzyme which can do the job, basically digest DNA at high salt concentration, we were scratching our heads and choosing between most commonly used expression host for enzyme or any protein of interest. So if you think about it, it's obviously bacterial host or yeast host. And again if we are making head to head comparison, if you express a protein inside of bacterial host, you need to break the cell open to license. Basically while using E host you can make protein secreted and make your job easier. Additional I would say advantage of using E host for protein production is definitely no endotoxin because if you express your protein in E coli then you have to make sure that your downstream process removes all of the endotoxin introduced by the outer membrane of the bacteria. However, one major advantage of making your protein expressing bacteria which actually made us to choose or tend us toward bacterial expression is no post translation modification. Because if one chooses air yeast host such as PKI pastoris for example, your protein of interest will be heavily post translate post translationally modified, especially glycosylated and as you can see here that's how it can look like. So here is like original protein like expressing bacteria, no glycosylation at all, very specific band in SDS page. However, if you would like to try some other enzyme during yeast then like that's how your product will look like very non defined molecular size or heavily post translation modified most probably coming from glycosylation. Now again we made a choice because we have really deep knowledge expertise in bacterial expression and remove of endotoxin and we continue further with that. And actually we were when we started to produce more commercial batches of this enzyme, we again very happy because batch to batch variability was minimal because again if you're expressing bacteria, you have the same band all on all, all and over again precise molecular weight. While again if we are comparing 2 competitor salt active nucleus on the market and again that's like what you're getting. And here some questions could be asked whether this post translation modification is batch to batch variable. First thing, the second thing, whether this post translation modification will affect batch to batch and zematic activity per units per milligram, let's say. And the third question already Sarah spoke nicely about it. If you think about the holistic AAV production process, at the end of the process when you have your AAV ready to be injected to the patient in need, you have to make sure that there's no of course DNA inside but also no protein, no endonuclease inside of your final formulation. And the gold standard for doing that, it's Eliza kit. And Eliza kit is based on antibody detection. In a perfect world you would have a sandwich Eliza using two antibody pair which basically binds to the epitope of interest and says whether there is, there is or there is no or under detectable level of your protein of interest. And it's very easy can be done if your enzyme of interest has very precise molecular size, like in this case. However, if you have heavily post translate enzyme, then again what exactly your antibody and Eliza kit will detect this band, This band or maybe something here. So then like you should introduce polyclonal antibody or make definitely make sure that you remove all of the species from your final dose. Again to summarize, what I was talking before is we choose the production of the coli because it results in clean and pure protein which is devoid of post translation modification and ensures high batch to batch reproducibility. OK, so we have our enzyme, we know that it has extremely high batch to batch producibility. But also from the literature and our previous knowledge, we know that endonucleases are heavily relying on the magnesium concentration and that's exactly as you can judge from this graph. What we tested, we tested concentration of magnesium inside of the buffer from zero to 10 milli molar and checked how it affects the enzymatic activity of standard endonuclease black dots at 150 milli molar as one can see without magnesium, no activity at all at 150 milli molar. Actually the range of magnesium that one can put in his license buffer can range from one to 10 milli molar because virtually it did not affect enzymatic activity. The same thing if you took take a look at our newborn salt tolerant benzonates at 500 millimolar. Again at 0 magnesium it's not active, but at 500 millimolar it again similarly active from one to 10 millimolar magnesium concentration in your buffer. Some problems may occur if you put standard and the nucleus at 500 millimolar NaCl concentration, which again it's not optimal at all, it's not digesting there or vice versa. If you put our newborn salt tolerant benzonates, we get 150 millimolar NaCl again. Then it's activity can be heavily affected by magnesium because the salt concentration of NaCl is not optimal for its activity. But a really optimal salt concentration from one to 10 millimolar magnesium, it's very safe range to use. Then we really wanted to ensure that our enzyme really does a job at 500 millimolar NCL and again using standard random nucleus. If you're taking a look at the X axis, it's different enzyme units per milliliters that we put in our surrogate DNA digestion procedure and here is like enzymatic activity. So standard endonuclease at 150 milli, molar concentration already as a concentration above 10 units per milliliter completely digested all of the DNA inside and we can say that, yeah, above that it's really all the DNA was digested. The same thing we can say about salt tolerant Benzomics at 500 milli molar and ACL. Again at the concentration more than 10 units per milliliter. It did the job exactly as standard and the nucleus does at 150 molar. And again if you put standard and the nucleus at high salt concentration, then of course you have some problems with activity because again standard and the nucleus is not active at high salt concentration and vice versa. If you put salt polar and Benzonas at low salt concentration, it's also not very active because again he was not optimized. So we can say that a range of magnesium ions is from one to 10 millimolar for each standard or salt tolerant benzonase enzyme and basically salt tolerant benzonase activity is enhanced by high salt concentration. In the next step, we were keen to know whether our salt tolerant benzonase is comparable to some other salt active endonucleases on the market. And what we did, we again digested the DNA at 500 millimolar or 1000 millimolar NaCl at one hour 37 degrees and check what's going on. So first of all, let's start with standard Aetna nucleus Again at 500 millimolar, you can see there's no full digestion as can be judged by the other gel. At 1000 millimolar there was no activity at all. Basically the DNA with or without enzyme is the same. Then if you're speaking about standard and nucleus, competitor did very good job at 500 millimolar NCA but also at 1000. But last but not least, definitely our newborn S tolerant benzonase has did the job equally well or even better. So it digested all of the DNA there at 500 or 1000 millimolar NCA. So again we can say that if they just DNA completely at 1000 millimolar NSCL, it shows similar or even better performance versus competitor A OK. Then we want to ensure even the strongest safety factor than there is in the market. Sarah already was talking about this FDA requirement for again for the AAV dose that goes to the patient in need should not contain or should contain less than 10 nanogram per dose of the DNA independently whether it's wholesale DNA or plasmid DNA. And the size of the DNA has to be below 200 basements. But we wanted to take this challenge and ensure that our salt tolerant benzonase at 500 millimolar makes even better jobs. So what you can see here we run the DNA at 4% agarose gel and using low molecular weight DNA marker, we again run the DNA without an enzyme as you can see it there at 150 millimolar assault tolerant benzonates. We know that it's not digesting the DNA completely. That's why we see some remaining of DNA around 100 base first. However, if you put the same enzyme at 500 millimolar NSCF, you see nothing even below 10 base per. So we can say say that like we consider even stronger safety factor and our salt tolerant benzonates at 500 millimolar, they just below 10 base per of DNA. So virtually already on the oligonucleotide side. Sorry, sorry. OK. Then we went one step further and thought, as Sarah already was presenting, when one needs to break the cell open, he needs to use detergents, right? Because otherwise the cell's cell membrane will keep in its contents inside. And we tested all four common detergents on the market, Triton X100 Polysorba 20 and also newly introduced detergents Divide and C 16 and Divide and 13 S 9. And we wanted to test the Zoe detergents when used with salt tolerant benzonase will not affect its activity. And again as you can see on this graph, So X axis tells you how much enzyme units per milliliter we put in the reaction and here's an enzyme activity as we judged by the presence of DNA. And our positive control was of course 500 millimolar without any detergent. And what we can say from this experiment that all of the four detergents if you put the salt tolerant benzonates enzyme above 10 units per milliliter at 500 millimolar and ACL did exactly the same. So we can conclude from that, but 0.5%, all of those detergents did not affect enzymatic activities. They did not inhibit it and just to make sure again to show it in the other way. So here we just quantify the DNA and here we wanted to run them on the agarose gel again in presence of Tritonix 100 polysorbic 20, odivarium 14 S 9 standard and the nucleus at 150 million molar makes a great a great job but as we know it digests all of the DNA. However, if you increase the salve to 500 millimolar, there's a lot of chunks of DNA remain across all of the different detergents. Vice versa, if you use salt tolerant benzonates AT-150 millimolar, again those chunks of DNA remain right. But if you use a salt tolerant benzonates at 500 millimolar, virtually of the old DNA is digested across all different detergents. All of the experiments that I was showing you up to this moment were done in kind of surrogate manner. So DNA was extracted from fish and like we did everything in very in vitro manner, like really to make sure that our enzyme is up to the specifications that we promised. However, we really wanted to make a real world test to produce Aavs and see how the new procedure effects its yields. And the first experiment was done using suspension hector and three and we produced AAV 5. And again once we already produce the AAV 5 we need to break the cells open and we broke the cell open using Tritonex 100 polysorbit 20, the Viron C16 and the Viron 13 S 9. And again another factor that we introduced we lyser cells at 150 millimolar or 500 millimolar using either standard tendonuclease or salt tolerant tensomates and as one can see across different conditions if we compare to the baseline. So our baseline number of capsules per milliliter as measured by Eliza was using 150 millimolar NaCl and polysorbate 20 license buffer with standard tend to replace And if we increase the concentration of salt in the lysis buffer and using salt tolerant benzonase and broke the cell open using polysorbate Triton, the Viron C16 of the Viron 13 S 9, we always saw an increase in capsids per milliliter as judged by Eliza from 16 to 39%. So again, we can say from that just merrily increasing the salt concentration for 150 millimolar to 500 millimolar, we won can expect increase in the tighter of Aavs, again as judged by Eliza as and as presented as capsid per milliliter. If one can say, yeah, I can increase my salt and get very nice AV titers using standard endomic lace, yes, the titers are there and they're also nice. However, just to remind you, if you break the cell open at 500 millimolar and using standard Endomer lace, one cannot remove the remaining DNA because it's still there and therefore the safety factor is gone. Again, kind of testimony. Right now the gold standard for quantifying the Aavs is either using ELISA kits for capsules per milliliter or digital PCR for genomic copies per milliliter. However, we wanted to walk an extra mile and to show that this procedure also shows nice results in the potency essence. And what do I mean by potency essay is we really wanted to detect that the expression level of protein of interest introduced by AAV in the endpoint essay is really increased because yeah, maybe I increase the number of capsids, but whether it's really affects the efficiency. Well, the next experiment was done in adherent Hectin and free cells and AAV two were produced. And what we saw in this experiment, again we did not quantify the capsids per milliliter, but we want, we really checked the infectivity, we checked the how many infectious units or transducing units per milliliter I have. And again our baseline was Lysis using 150 millimolar of NSC and polysorbate 20. But if we compared all different conditions, we're just increasing the salt concentration to 500 millimolar and using polysorbate 20 or lysing even without the detergent. We also tried that or even try, sorry, lysing using Triton X100 or Divided 14 S 9. We always saw, I'm not, yeah, that's not a mistake. We always saw at least tenfold increase in infectivity from 1700 to 2200 percentage because it's a log scale. And that already tells you, yes, we can maybe increase by 10 of percent the AV production efficiency in capsules per milliliter. But in our experiments in our hands, the infectivity was improved at least 10 times and again we always as a high salt concentration. One would like to use salt tolerant benzonase again if you will, standard and the nucleus for digestion and high salt concentration. You still stay with a lot of DNA which is not digested coming from the plasmid coming from the whole cell DNA again. We actually do not have a complete mechanism to explain such a nice boost in infectivity. We have a working hypothesis, but just to summarize the things that I was telling you before. We can ensure that using salt chlorine benzonase, it digests DNA at high salt concentration to very low base personals, DNA below 10 base person in size. And also we can ensure process efficiency because high salt concentration, again that's our working hypothesis hinders AV aggregation. So now the high salt AV cannot stack to each other anymore. They are really in the single molecules or moieties and by that we can also, using high salt, ensure removal of DNA from viral capsids. At high salt, the DNA is removed from the capsids. We also ensure that the AAV is not aggregated anymore. And now basically when your Aavs are freed from DNA and aggregation, they are much more infectious and easier to purify into further steps. With that, I would like to hand over the presentation back to Sarah. Before we go back to Sarah, thank you. And Dmitry, thank you for your presentation. We would like to ask you our next call question. The question is would you consider at this point high salt license in your process, yes or no? You have a few seconds now to choose your response. OK. The poll is now closed. Thank you for sharing your insights once again. We truly appreciate your active participation. We now go back to Sarah to continue her part of the presentation. Sarah, thank you very much. So, OK, we've seen some very nice application data to kind of show that high salt actually has an impact on the process yield. No, the question is, OK, let's get back to basics writers. When do I need to use what? No, we're looking at an entire world of nucleases, right, with different claims, different condition of operations. And this is definitely what we're saying and what we would recommend whenever your saw concentration will be above 200 millimolar, go for salt tolerance because definitely you're going to see an increased DNA digestion ability. And also let's get back to that, right. If you look at this purple box, you see that at 500 millimolar, 29% tighter increase and close to 2000% infectivity increase. This is definitely something that's very appealing. So to finish on on that point about the DNA digestion itself, always look for the efficacy of the digestion in base pairs, right. Don't look just that activity as a kind of nebulous thing. In the end, what you want to know is can the nucleus achieve very small DNA fragments? That's the question. So if we summarize what was said, so White choosing new benzonates, right, especially if you're already using benzonates or maybe all the type of nucleases. So we've seen increased types or we've seen increased infectivity. Now if we look at the enzyme itself, having an enzyme with a high homogeneity from batch to batch and devoid of post translational modifications is definitely also something that is very key. DNA digestion below 10 based pair is also something key. So all those points are very something to consider when you're looking out for enzymes. So we will close progressively this presentation, but before we go a couple of points I would like to cover. The first thing is that we're talking about the Benzonase because it's a strong name that a lot of you know and this brand is evolving, right. So we are bringing a new member to life, a new member dedicated to high salt applications. But also what we would like to share with you is that the Benzonase is actually one of the nicest success story of the life science industry. And why does that matter? It matters when we're talking about supply robustness and to make sure that whenever you need the product you have it. So the Benzonase actually has redundant production sites, which means that we have several sites to make Benzonase, which means that they should be no issue if ever one of the sites should shut down. So that's very important. That means also that thanks to that strategy, we've been able to absorb and need multiply by 5 in couple of years, especially during COVID. There was no issue with supply chain. We have the same supply robustness for or Eliza kits, right. So here again we're done in production sites. So what we're bringing also now to this portfolio is customization. So if you have any needs and when it comes to packaging specifications that would not be already on the COA, let us know, right. So when you consider new clays, again look at it from a more holistic point of view, supply robustness and any kind of other claims that are definitely highly regarded by regulators. So now I would like to guide you through the specifications of that new enzyme, if you're familiar with the standard benzonase that we offer or maybe the benzonase safety plus. So this new salt tolerant benzonase is going to be a sort of hybrid, it's going to be non animal origin, that's a given. It going to have a lot of the safety plus claims like terrogate samples, mycoplasma test temperature strips. The difference is going to be that this advantageous virus test is going to be on demand. So it's going to be like one of those custom possibilities that you have. So yes, new benzonase will be launched soon. We have samples already available of GMP grade material and it's going to be non animal origin. So because we've seen at the beginning right that when we're talking about DNA digestion, actually the topic is, is far broader than this. It's about like Cellulitis, DNA digestion. Sorry and purification afterwards. We have a network right of experts that can help here for technical consultations for process optimization. So let us know if we can help you in any way. We have also a lot of expertise with AAV manufacturing. We have our own sites for AAV manufacturing, so do not hesitate to leverage this network that we have. So this new enzyme will also come with all the pack size that already exists, right? So 5 million units, 500K units and so on. Really the message would be the product's going to be there very soon. If you want to anticipate regarding qualifications, proof of concept and so on, get in touch because we have to material at hand and a word as well on detection, right. We've discussed with DEEMA how accurate detection is needed and how difficult it can be based on where your enzyme is coming from. So we are very also happy to say that analyze that it is of course joining also the benzonase family for this new salt tolerance enzyme. Don't be fooled on the market there is no Eliza kit that can detect this enzyme for the very simple reason that this is not available yet commercially. So if you say something that's supposed to be detecting it, this cannot be true. So we will have this kit very soon as well. So here again, you know if there is an interest, let us know. So I'll finish talking about the M proof program because our Benzonase is definitely connected to M Proof. So for those who are not familiar with it, Merck has a documentation program that is helping manufacturers for qualification risk assessment. This program is a sort of kind of dossier management program where you find everything online and you do not have to ask for single documents. So we've improved, you have different grades that we will see here and depending on your application, depending on where you're using a specific raw material or specific consumable, you will have different number of documentation available. So we are very happy to say that the Benzonase is part of Emperor of Experts, which is one of our highest quality grade GMP and that means that all those dossiers that you see on the left are available. So stability studies, manufacturing information, all of that is available in a single location. So really I would say thanks very much for, for being with us today. Let's just summarize what we've discussed in that in a couple of points. I think the key message here is that salt is definitely needed to increase AAV yields and we've been seeing it. So this is something that has not been highly used by the industry so far. We believe this is because the nucleus ability to digest that high salt was one of the bottleneck. We hope that we are lifting this bottleneck which means that we are expecting the industry to be using more and more high salt license to increase process yields. This new Penzonase is compatible with all detergents, all standard detergent. So no concerns here. It is suitable for AV production comes with really exhaustive claims right far beyond GMP claims and this product will also have this redundant production site strategy. So very excited to to bring that to life and again samples are available if needed. So thank you very much. And I guess just a little disclaimer about all the colleagues that managed especially those R&D experiments and though I will open up the the Q&A session. Thank you so much, Sarah. OK, now it's time to answer a few questions that have come in already from our audience. But before we do, I would like to remind you that it's still not too late to send us your questions using the QA widget, which Sarah kindly mentioned. We will try to get through all of them, but if we run out of time, we will respond to you individually. 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 Sarah who will start answering questions that have come in so far. Thank you. So there is a first question that came in before the webinar. So I'm already using a salt active nuclease. Do you confirm the immune quantification could be inaccurate with yeast Express protein? I'm currently using an ELISA kit, so maybe GMA, I can turn that question to you. Yeah, basically that's brings me to the slide that I was showing kind of protein expressed by yeast or protein expressed by bacteria. So if someone expressed by yeast, you have a lot of protein sugar interactions and one you immunize, we're using those protein, your antibodies most probably kind of detecting this sugar protein moieties and not detecting the whole protein. So if you express yourself active nucleus in east, then like your ELISA kit can give you some problematic results and you don't know which species exactly you are detecting or like what exactly was left after your downstream process purification. Yeah, because that can be really problematic. Thanks Timo. I think maybe another question that is quite, quite interesting as well. So someone that's using high supplies and buffer exchange. So then that means theoretically if you make the buffer exchange step, then you can use any kind of nuclease. And so is that making a difference then the buffer exchange step or or or what do you think? Yeah. So here is exactly the kind of issue that we were trying to tackle. If you think about it, at 150 millimolar, there's still a lot of DNA protein interaction. And what's happening is one who breaks the cell open DNA or even chromatin DNA is really sticky to the capsid nature. So the only way that you can really ensure that all of your DNA is a plasma DNA, wholesale DNA or chromatin pieces are removed from the viral capsid is to increase the concentration of the cell to 500 millimodar or even higher. However, like if you increase the concentration and using standard endodo plates, then your enzyme is not digesting it. So but if you again, let's say increase to 500 millimodar and then decrease it back again again by buffer exchange, all of the unremoved DNA potentially can bind back to your Aavs. So like you have to make sure that you're using at the same time high salt, removing all of the impurities from your capsids and using the enzyme that can work as a high salt concentration. Thanks, Tima. I can take the the next question. Is Merck planning to stop using this starch aluminum tubes? So the answer is yes, definitely. We're looking into much more sustainable packagings, so for that product but also for all or enzymes. So stay tuned, it's coming. We have a question also how to deactivate the Saltolin Benzonase. So Dima, yeah again one definitely have can choose a lot of ways, definitely some heat and activation. But yeah, by that you can also inactivate your Aavs as I was showing you in some data that like if there's no magnesium ions in your buffer then the enzyme is not active. So by that, just adding simple EDTA or similar chelator of magnesium ions will do the job by inactivating the enzyme, yes. I would add to that also that enzymes are removed quite easily during purification steps with TFF chromatography. So this is definitely not or should not be a concern. I can take the next question as well. Are you recommending high salt buffer in New York detergent or in addition to it here we're talking in addition? Sorry, we've seen some cases where there is like just use of high salts for to achieve the the lysis and no detergents for it. We observe the difference in India in deficiency of the of the cell lysis. So here we're really talking both together. So demo, So for you do you recommend using detergent with more purified proteins, viruses, You mean detergent of the the viral detergent or any other no, like the the salt tolerant benzonase like can can it be used for other applications? I guess that's the question from the moment the salt concentration is is high, but I think the answer is yes. Oh, but no, it's detergent when you're right. I don't know actually, I don't know if you could clarify the question because whether it's detergent, there is no issue between detergents and salt concentration, right? No cross reactivity. And for the enzyme, then yes, the different applications from the moment they are at high salt would make sense. Sorry I did not catch that. So that one also for UDIMA do we add high salt prior to benzonase or after benzonase DNA digestion? What is incubation time for benzonase and their high salt? Yeah, so definitely to achieve the best results, again I will just recommend to lyser cells or have a lysis buffers already containing 500 millimod on ACL your detergent of choice which was suitable for your process optimization and just salt tolerant benzene SO500 millimolar and ACL 0.5% of your detergent and salt tolerant benzonase. And in all of our AV experiments we digested the samples for two hours, so lyser cells for two hours. So basically both lyses and digestion of DNA. In previous surrogate experiments we incubated even for 30 minutes that was perfectly enough. So but again, it's really depends on your process optimization and one should definitely optimize what is a needed amount of enzyme and what is the time to ensure proper lysis and full digestion of your DNA. OK, perfect. I do not see more questions at this time. So I think we can close the session. Yeah, that's great. That's all the time we have for today as well. Thank you very much for all the questions. If we didn't get to, if you didn't get to your question, please feel free to get in touch with your point of contact in our organization. To register for future webinars or to access our archive webinar library, please visit our website. I would like to thank Sara Lucia and Dimitri Zabuzenski for today's presentation and thank you to your audience for joining us. Have a great day everyone and stay curious. Thank you. Thank you. _1732521724435

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