Hi everyone and welcome. My name is Sonia and I will be your moderator today. Thank you for joining today's webinar key benefits of PCR based mRNA manufacturing for clinical development. 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 Dr. Julian Moshegeti, Strategic Marketing Manager for mRNA Solutions. In his current role, Julian is responsible for the strategy, development and positioning of our mRNA CDMO offering. He holds a PhD in Translational Immunology from Heidelbeck University and Prior to joining our company, he gained extensive experience in translational research with strong expertise in the areas of novel modalities and in vivo gene editing. Here with us today is also Dr. Ike used Principal Scientist for mRNA innovation. ICO holds a PhD in Cellular Biochemistry from Frankfurt University and has brought technical expertise in synthetic biology and molecular biology. He's Principal Scientist for innovations and technologies in our mRNA service offering. Prior to joining our company, he was responsible for the development of the upscaled mRNA manufacturing process and implementation of M Tech technologies in Rd. Before turning things over to our presenters, I'd like to just cover a few housekeeping items. At the bottom of your screen, there are multiple application widgets that you can use. All the widgets are resizable and movable, so feel free to move them around to your liking. If anything stands out to you during the presentation, please give a presenter's feedback by using the Reaction widget. You can expand your slide area or maximize it to full screen by clicking on the arrows in the top right corner of the slides. If you have any questions during the webinar, you can submit them through the Q&A widget. We will try to answer them during the webinar, but if a more detailed answer is needed or if we run out of time, we will answer later via e-mail. Please know that we do capture all the questions. You will also have the opportunity to participate in a quick pull question throughout the session. I encourage you to take part in this. If you're watching this on demand, you can still submit the poll responses as well as sending in questions. The webinar is being streamed through your computer, so there's no dial in number for the best audio quality. Please ensure your computer, speakers or headset are turned on and the volume is up so you can hear the presenters well. An ondemand version of the webinars will be made available afterwards and can be accessed using the same link that you've already received. So that's it from my side. It's my pleasure to turn things over to our speakers. Julian, over to you. So yeah, thank you for the kind introduction, Sonya. I'd like to also extend a very warm welcome from my side and I look forward to sharing some really interesting insights on our PCR technology and then also at the end, engaging with you in the Q&A session. So the life science business of our company Merck KGA, Darmstadt, Germany operates as Millipo Sigma in the US and Canada. And our company was actually found in 1660 and it was oldest pharmaceutical and chemical company still operating. And really building on this continuity and also really long track record, we are also continuously expanding our CTDMO services along the full mRNA value chain into an integrated offering for the benefit of our clients and ultimately the patients that they serve. So for today's web enough we would like to kick off things firstly. With a brief overview on the developments and trends within the rapidly evolving M and A market landscape and how we as a service partner enable the success of these programs through our offering. And then secondly, we'll follow up by a deep dive on how our unique PCR based M and a manufacturing workflow can actually accelerate the development and also manufacturing of M and a drug substance. So for those of you. We've watched our webinars in the years past. You should already be a bit familiar with our brief overview on the market growth. So as we can see from the Mr. and a pipeline evolution graph shown on the left hand side, Mr. and A has actually already been researched for many other indications before the COVID-19 pandemic hits in the beginning of 2020. However, as you can imagine, this approval of the Mr. and A based vaccines have significantly driven interest. In Mr. and a as a modality due to this very successful first proof of concept leading to a huge increase in the number of preclinical but also clinical programs since February 2020. So really to discuss some of the numbers, in February 2020 there were around 127 programs in total for Mr. and a a year later in February 21, there were already 200 programs. In February 22, there were already 349 programs and then skipping forward to February of this year, they were already more than 572 programs in the full pipeline. And while the focus of Mia based COVID-19 vaccines have normalized, even today we see a super strong growth in the overall Mia market really fueled by non COVID indications. I would like to draw your attention now to the right hand side. So of those 572 programs already 172 have progressed to clinical trials with the compound communality by Byron Tech slash Pfizer and also Spike wax by Moderna having received full market approval on the international level and this is a really important fact. The majority with around 77% of all programs today are actually focused on non COVID indications such as other infectious diseases, but also on applications in the field of oncology and a multitude of other indications as well. And in particular those other indications really underline the versatility and promise of mRNA that no other modality can match to date. So this slide should really provide you a very brief overview on our integrated M RNA CTDMO capabilities to accelerate your programs by really providing solutions from the M RNA drug substance all the way to the final drug product. And we as a company really believe in M RNA as a breakthrough technology. And that we're only seeing the beginning of its historical success. And really as such, our key focus and strategy is to bring the experts for each individual business and pillar into our globally connected mRNA LMP network to really streamline the journey and allowing scaling of a project from preclinical to commercial all with one single partner. We have more than 25 years of experience in the development and manufacturing of lipids for drug delivery. In 2021, we expanded towards MRA drug substance manufacturing through the acquisition of Antech, again a leading MRACDMO with more than 15 years of experience in RNA manufacturing. More recently, we expanded also through the acquisition of our Indianapolis site. Commonly known as X lead, whose capabilities and more than 20 years of experience again complement the existing offering through LNP formulation and final fill and finish all the way to the drug product. And most recently we actually launched an early formulation screening service out of our headquarters in Darmstadt in Germany. So really. With our experience sites, we are building a globally integrated offering where we can significantly decrease supply chain complexities through harmonized processes and really enhance our clients speed to market, which again and most importantly will in turn really accelerate the access to life enhancing therapeutics for patients. So really for today's webinar let's take a deep dive into our M and a drug substance manufacturing service. So as mentioned in 2021 we acquired Amtech, a highly experienced M and a CDMO based in Hamburg. And Amtech had this had developed this really unique PCR based workflow for next generation M and a manufacturing that really eliminates a lot of the complexities known and associated with the conventional M and A manufacturing process. And really excitingly, we've successfully solved the challenge to actually scale up this technology and are launching our M RNA Center of Excellence in September of this year. So should you be looking for a partner in M RNA, feel free to reach out already today to secure your slot and our M RNA Center of Excellence will enable us to provide M RNA at all scales and qualities. For all stages of M and a programs lifecycle from preclinical to commercial demands. So through this M and a center of excellence, we provide flexible capacity as a developer including in house process and analytical teams. And we are also continuously improving our offering by our by our R&D innovation teams that we also have in place and Ike is one of the members there. So for preclinical applications we have PAD and also non GMP labs available and illustrated by the tiny map in the bottom left. Our mRNA center of excellence has two harmonized GMP manufacturing sites in Hamburg and in Darmstadt allowing for small scale to large scale manufacturing and as mentioned before including for late phase commercial manufacturing all from a single source. So as a CDMO, we have also established a fully scalable GMPM on a manufacturing platform that really allows for the flexibility that is needed to adapt to each client's individual requirements or even also allows for rapid tech transfer in of established clients processes. And this platform includes fully scalable unit operations like TFF and other chromatography based methods including affinity purification. And also here my colleague, I will briefly expand on this process in just a moment. Since our manufacturing platform can be used with our next generation PCR based manufacturing process, I just very briefly wanted to highlight some of the key advantages of the technology. So first of all, our PCR based manufacturing approach can really significantly deep bottleneck. The critical plasma DNA raw material as our PCR based approach requires approximately 10,000 fold less plasma DNA to generate the same amount of M RNA when compared to the conventional plasma DNA linearization approach. And also these upfront cost and time savings are further emphasized because we only need super small quantities of plasma DNA to begin with and we can perform simple. Plasma banking and we may not even necessitate master sell bank establishment at an early stage, again saving months and hundreds of thousands in early development. Furthermore, the plasma DNA requirements only according to GMP principles and not full GMP since the IVT template is actually generated through the PCR step and not a single piece or constituent of the plasma DNA actually goes into the IVT step. In contrast to the classical linearization approach. So in addition, our process is also very flexible and can be very easily adapted for variance, allowing for instance for flexible introduction of a fully synthetic Poly a tail template directly during the PCR step itself, again circumventing the need to have the Poly a tail encoded in the plasmid DNA. And therefore also mitigating the risk of obtaining truncated Poly a tail templates in the Mr. and A. Furthermore, this really also again obviates some of the manufacturing challenges that arise for the manufacturing of plasma DNA encoding such long homopolymeric sequences. Our Mr. and A manufacturing method really also leads to very consistent and reproducible results from around 200 up to 9000 nucleotides. But longer contracts such as self amplifying RNAs have also been very successfully manufactured. But as with any process, these always require custom analytical reference, standard development and validation. So to ensure high quality of the MI A, the homogeneous PCR based IVT template is synthesized in a very tightly controlled in vitro reaction using high fidelity polymerases. Again, obviating the need for plasma DNA linearization and therefore also circumventing further risks of obtaining truncated or trailing products generated by not completely linearized plasma DNA. And lastly, by design, our technology has a direct impact on the purity of the MNA itself right from the beginning of the process already in the upstream part. As the IVT template is generated in the controlled completely cell free in vitro process that requires 10,000 less plasma DNA, it also equally mitigates the risk of carrying forward microbial DNA, microbial protein and also endotoxins. So with this I would like to conclude my section but before I hand over to my colleague Ike. We would love to hear from you in form of a poll question. So the question that we would like to ask you would be at what stage is your most advanced mRNA program And the first option A would be currently not invested in mRNA yet, but looking into applying mRNA technology. Option B would be we are at a discovery stage, option C. Would be we are in a preclinical stage for instance performing non human primate studies. Option D to F would be within clinical phase one, two or three respectively. And lastly in G our product is already marked. So I'll give you a bit more time to think about the answer and select the right one and I'll just repeat the question and the answers one more time. So at what stage is your most advanced MMI program? A looking into applying MI net technology, B discovery C preclinical D to F would be clinical phase one to three and G would be finally marketed. Very interesting. Thank you for sharing those insights with us. It is much appreciated. And with this over to you I can very interesting. And yeah, thank you Julian for demonstrating how our integrated offering is meeting the demands of this extremely growing market. And now also one welcome from my side. It's now my pleasure to share with you some of our technical highlights of our PCR based mRNA manufacturing process. Our mRNA manufacturing process can be divided into three major phases. In the first phase, we make the mRNA. Here our process starts with a specific amplification of the gene of interest by PCR and in direct contrast others here start with an enzymatic linearization of a large pleasant batch. In our second and third phase, we include dedicated units for purification and filling of the final mRNA drug substance and in total we designed this whole process to provide. A maximum of flexibility and scalability. For example, the process also enables cool as well as post translational capping. In parallel to this high flexibility and scalability, we also offer high quality attributes for the final Mrnas. For example, we manufacture Mrnas with a strictly defined size with a minimum of residuals and with pulley a tails above the range of processes. That are limited by using the linearized plasmids and the recombination events in Ecola. And the key to this broad spectrum of features is the PCR based generation of IVT templates. And for this purpose we developed a reproducible PCR setup that enables a strong as well as specific amplification. And on the left side, you can see the strong and specific amplification in Agro's gel electrophoresis and there you can see this strong and clear product lens. On the right side you see the high reproducibility in capillary electrophoresis. Here we show the overlay or mean value of 20 individual group PCR reactions and all of them together exhibit a very distinct peak at the expected product size. To ensure a strong amplification with high fidelity, we analyzed our PCR based IBT templates with next generation Sequencing and on the left side you can see three individual model sequences we used for such an assessment and in this case we used a 2004 thousand and 6000 base per reference. And on the right side, you see the details of the corresponding next generation sequencing analyzers. And by sequencing the same position several 1000 times, we ensured a high coverage and high quality sequencing data. And to further ensure an accurate analysis, we also restricted the whole assessment to high quality sequencing data. To this end, we used a thread quality score of at least 30. To guarantee a base call accuracy of at least 99.9% and based on this data and based on established procedures, we could determine error rates in the range of 10 to the power of minus 9 to 10 to the power of minus 10. And these error rates are actually in the same range as E coli replication which forms for sure the origin of plasma based IVT templates. And just as another contrast here, it's actually more likely that you win in the German lottery than to generate the mutation in our PCR setup. And encouraged by these results, we also developed a second optimized PCR protocol and again analyzed the PCR product fidelity by next generation sequencing and applied again the same high quality standards. And as you can see here, we were able to maintain this high fidelity while in parallel significantly increasing the PCR yield. Next, we would like to focus on the purity profile of our PCR based IBT templates, here in this case with an E coli protein specific ELISA. And for our PCR based templates, as you can see here, we could not detect any kind of residual E coli proteins in several batches. However, in a direct contrast for purified blastnets we obtained externally, we found significant amounts of residual E coli protein and these are proteins that you may carry over into your IVT reaction. Next, we would like to focus on the high reproducibility. Of mRNA synthesis based on these PCR based templates and on the left side you see three independent batches of crude PCR based IBT templates in capillary electrophrases and on the right side you see the corresponding final mrnas. And strikingly, this definite size of the PCR product is reflected in the final mRNA. To further analyze the impact of our PCR based process, we contrasted our mRNA and competitor mRNA generated via delinearized plasma processes. And for an appropriate comparison we used here Mrna's that were downstream manufactured by a simple silica membrane process. And on the left side you can see that our PCR based upstream process resulted here in a clearly defined product. At the expected product size and in direct contrast for the competitor M RNA, which was manufactured using the linearized plasmids, we detected several undefined signals. To obtain here more comprehensive overview, we actually analyzed several competitor M RNAs, all of them manufactured by linearized plasmids and. Apart from several undefined signals, we could also detect a significant difference between the expected product size and the actually measured size. And for more detailed robust analysis, we contrasted directly the size deviation of our mrnas with competitor mrnas, again in capillary electrophrases. And for our PCR based M Rna's, we detected nearly 100% of the expected sizes. However, for competitor M Rna's generated via linearized plasmids, we detected A heterogeneous distribution and the size deviation of approximately 16%. And within the clinical study, this heterogeneous size distribution may affect the observed performance of your M RNA. And finally, we would like again to focus on the purity profile of our mrnas, again with an E coli protein in specific ELISA. And when we just look at the mrnas for our PCR based mrnas here, we could not detect any kind of residual E coli proteins in the final mRNA products, however for some competitor mrnas. Also manufactured using linearized plasmids, we detected significant amounts of residual E coli protein even in the final mRNA product and this in some brings us back to the quality of our mrnas. And it further demonstrates that our PCR based IVT templates offer a broad range of advantages from scalability to a distinct product size and towards an improved purity profile. And with this, I would like to close the presentation with three major takeaways. First, our PCR based mRNA manufacturing process robustly generates Mrna's with a reproducible performance. Second, our process design itself allows to provide Mrna's for early clinical as well As for late stage or commercial needs and 3rd. As an end to end service, we provide unique and integrated solutions for your mRNA products. And now finally, I would like to thank all of you for attention and I hand over to Sonia for the QA session and I'm looking forward to your questions. Thank you Julian and Iker for sharing your insights. Like I said, it's time to answer a few questions that have come in from you. But before we jump into the Q&A session, I would just quickly like to remind you that you can still submit your questions using the Q&A widget. And as a last reminder, this webinar will be available on our website soon and all participants will receive an e-mail notification when it is available for on demand viewing. Now I will look at the first questions and hand it back to our speakers for answering. So Julian, actually there have been several questions relating to manufacturability of longer mRNA using the PCR technology and potential challenges with that. Can you comment on that a little bit? Yeah, generally a super relevant question in particular as the field is also looking towards longer RNA constructs I e.g. including CRISPR or even self amplifying RNAs. So in principle I would separate the question into two distinct parts. So the first one really relating to our PCR based technology itself. So as I briefly alluded to, we have successfully manufactured. Longer RN A's on a routine basis up to around 9000 nucleotides including and for instance encoding for CRISPR and within the Mia sequence itself. But we've also actually manufactured self amplifying MI A's in the range of around up to 13,000 bases long. In principle these PCR based IBT template generation method is also very suitable for even longer MI A's, so that in itself is not a limitation. More importantly is really when dealing with such longer MRA sequences is the manufacturing itself and that relates to any MRA manufacturing technology applied. The challenges really come then in terms of viscosity challenges during the IBT itself and also of course the subsequent downstream processing. So I think that is the general truth that is important for longer Mr. A's. And as alluded to beforehand as well, longer contracts larger than 9000 KB require actually custom analytical reference, standard development and validation. So I hope that answers the question. Thanks Judy. And I think that was very helpful. I think the next one goes to you. Is the downstream part of the PCR process different to other conventional approaches? Yes. Thanks for this question. This is actually a very, very good one and it further highlights the differences of our process here. So our upstream process is for sure highly different since we use the PCR based generation of IBT templates and as Julian already mentioned since we. Start with the PCR based template. We have a lower amount of residuals, but for the downstream process itself that comes. Let's define it immediately after the IVT reaction. There's no major difference. The biggest difference is that we have for sure to get rid of a lower amount of residuals in contrast to others, but in theory this process is also suitable. To manufacture any kind of existing mrnas. So after the IBT reaction, this process is suitable for anything for any mrnas. Okay, thank you very much. Back to you, Sonia, I think we actually have another one for you. You did mention that the PCR process allows flexible polier tailing. What are the differences in contrast to plasmid encoded polyatails? So let's make a sort experiment, right? So imagine you find a literature that it may have to increase the expression of your protein based on your mRNA when you fine tune the polyatail itself. And when you would rely on the plasma based process, you would start with let's say 5 constructs and you order this plasmas and in parallel. When you're using PCR based process, you would start with five primers and guess what you get earlier, This will be the primers for sure. And then you see well one of these constructs is behaving well. Let's fine tune this a bit more and make a second generation. And then you order new plasmids or you order new primers. And when you get the second generation of plasmids, maybe this takes your time and then it's six months or even longer for the primers. You might be done within three months just trying this third one. And for us, we are even able to do this in a clinical scale or even later. So you can always kind of change the primer without much effort. But if you would need to change the whole plasmid, this would take a lot of money and a lot of time. Yes, I think, I hope this answers the question. And back to you Sonia, I believe so, yes, thanks Julian. The next one I think is for you. So do we accept customer pre-ordered or onsite visit before we start actual mRNA production for clients? That is a very specific question, but yes, we actually do that. And this is also one of the really cool parts about the two new GMP manufacturing sites that we've built up. So the one in Hamburg and the one in Darmstadt, they're actually designed in a way that. The manufacturing suites themselves can be inspected or visited from the outside. So there's a lot of glass and plexiglass going into it and you can actually look into the different units and manufacturing slots without actually having to enter the clean room site itself. So that's definitely something we can do also during the course of executing your specific project. Alluding also to this point, since we're in the process of launching our Amani Center of Excellence, should you at any time be in principle interested to visit one of the sites and we continue to engage along the journey there. Even a site visit is also something we can do before kicking off the project. So if that is of interest, please do get in touch. Back to you Sonia. Okay, thank you. We do have quite a few more questions, so I'm going to read out the next two. Subsequently, is the Poly a sequence introduced by primers? And how to make sure the PCR reaction only generate targeted sequence? Julian, I think this belongs rather to you, but correct me if I'm wrong. Yeah, either way would work I'm sure so. The first question was is the Poly a tail introduced through a primer and the short answer is yes, in the simplest form, yes. The second part is how do you ensure that a PCR only does a specific amplification of the sequence that you have in there. And again, this is the beauty of not using genomic DNA as an amplification source. You're using a super defined plasma DNA backbone and the primers that we use for the specific insert amplification are so well tailored that there is absolutely zero chance of generating any type of off target amplification in that, right? Because it's a very defined sequence. So you get a very clear and distinct amplification band of the insert and that again will then serve as your IVT template. I hope that. Answers both questions. Back to you, Sonia. Yes, thank you. How about commercial mRNA vaccine product scales? Would PCR still be suitable for batch sizes beyond clinical phases? I could you want to comment on that one. Thanks for this question also very, very good question. So. The short answer is yes, yes for sure. And this was a challenge and I think we solved it. So the whole process design is really that the process is harmonized for these lower quantities up to this really high commercial quantities. And it's always the same process, always with the same quantity. And since I was involved in the development, I can tell you this, it was not easy, but we solved it. And yes, I hope this answers the question. And back to you, Sonia. OK. Thanks. I think we have time for one more question. Let's quickly have a look. Yes, could you support and supply PCR amplified DNA in the gram or kilogram, gram scale for its clients? Anyone would like to, if you want, I can take up this question. Sure. I'll take that question. So it's basically asking if we would also already just apply the PCR generated IVT template if I remember correctly. So in principle the scale up of our technology would definitely enable that, but our operating model would be then to really provide the final purified to decline itself and not necessary already sell the IVT template to a client, but again. If that would be a need and it would be a significant opportunity, it could in principle be discussed. I hope that still is satisfactory answer to your question. So yeah, with this, back to you, Sonya, Perfect. And I think with this we do have to close out. So thank you very much for the interactive session. If we did not get to your question like mentioned earlier, we will respond via e-mail. I would like again to thank you Julian and Ike for today's presentation. It was super interesting for the audience to future register for webinars or to access our webinar library, please visit our website and you can also register for the 2nd webinar of our mRNA series who counts as an expert establishing analytical methods for emerging mRNA based therapies by. By clicking on the image in the take action widget which should be on the bottom right of your screen. Thanks again for joining us and have a great rest of your day. _1732475850493

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