Hi everyone and welcome. My name is Mackenzie Landgraf and I will serve as your moderator today. Thank you for joining us for today's webinar who counts as an expert establishing analytical methods for emerging mRNA based therapies. As your moderator, it is my role to ensure that we make the most of your time with us. I'm here today with Amy Glackis and Martin Ducheco. Amy is a global Business lead for NGS and novel modalities with over 15 years of experience working on various viral vector mRNA and CRISPR projects. She provides technical and consultative support to organizations working with RNA vaccines and therapeutics. Martin is the Technology lead for product Characterization, overseeing the development of new analytical testing services. Martin has over 12 years of experience developing methods for the characterization of novel biotherapeutics. I'm excited for our presentations today, but before we get started, just a few housekeeping items. At the bottom of your screen are multiple application widgets that you can use. All of the widgets are resizable and movable, so feel free to move them around and get the most of your desktop space. You can expand the slide area or maximize it to full screen by clicking on the arrows at the top right corner. If you have any questions during the webinar, please submit them through the Q&A widget. We'll try to answer these during the webinar, but if a more detailed answer is needed or if we run out of time, we'll answer it later via e-mail. But just know that we do capture all questions. You'll also have the opportunity to participate in a quick poll question during the session, and I encourage you to take part. If you're watching this on demand, you can still submit a response and you can also find our new reaction button. Just look for that little thumbs up emoji that allows you to give us some immediate feedback on the presentations, topics, or anything that stands out. This webinar is being streamed through your computer, so there's no dial in number. For the best audio quality, please make sure your computer, speakers or headset are turned on and your volume is up so you can hear our presenters. And lastly, an on demand version of the webinar will be available after and can be accessed through the same link that was sent to you earlier. All right, that is it from my side. Amy, I will turn it over to you. Thank you Mackenzie. I'm going to start by talking about analytical requirements for MRNALNP products. mRNA as a modality has emerged as a safe and effective means to reduce the spread of infectious disease and minimize symptoms when a patient is infected. The numerous promising applications for mRNA is is a therapeutic have received much more attention as a result of these heroic advancements. We're excited about the prospect of mRNA being used as a prophylactic vaccine, therapeutic vaccines, therapeutics and in cell and gene therapies and its potential to treat indications ranging from influenza to Stargardt disease. mRNA is a therapeutic contains an open reading frames by untranslated regions and a five prime cap and Poly a tail at the three prime end of the molecule. It is often, but not always, encapsulated in lipid nanoparticles or LNP's. This is special because the formulation of the drug substance is unique from the formulation of the drug product. This requires a unique set of tests and specialties for both DS and formulated DP, which I'll touch on a little bit more later. I think that we all recognize that given the novelty in the varied applications of mRNA. Is extremely important that the quality control testing strategy is supported by proven experts. You may not find more than a handful of people that have supported testing for the release of a commercial mRNA product. However, if you can find organizations that are consultative and provide analytical development and characterization that fit into your specific needs, you're off to a good start. It is also ideal when you find a company that is able to provide phase appropriate validation. GMP testing and release instability of your drug substance and your drug product all within a single organization. One of the ways that we try to lower the burden to having a phase appropriate method is through a combination of platform to GMP methods and the development of product specific methods. I'll start with platform methods. Whenever possible, our scientists will develop a validated method that can be applied to a molecule type like mRNA and be performed under GMP conditions regardless of the sample matrix. This efficient approach allows our partners to save development and validation time and allows for a more economical start to their program. On the other hand, we all recognize that this does not work for every critical quality attribute and product specific development will be necessary. In these cases, we will either transfer in a customer method or start development of a new method, undergo validation readiness and then perform phase appropriate validation. Further, we sometimes will have a combination of both approaches. In these cases, we'll conduct A portion of a method development and then move forward with finalization of the method and validation on a molecule to molecule basis. Imagine you're already thinking about which methods you would consider falling into each of these categories. Our experience and expertise has really allowed us to have an understanding of what an mRNA testing portfolio should look like. If you're listening to this webinar, you probably already have a good idea that it isn't as simple as just looking at testing mRNA. Rather, we need to look at complex starting materials like plasma DNA. Unformulated mRNA, the drug substance and the drug product, which in many cases is the mRNA formulated into the delivery system. Commonly, LNP's, the critical quality attributes, or CQA's are the same from each of these types of starting materials to the next. However, we would not necessarily be able to use the same set of methods to look at plasma DN, AM, RNA&LNPMRNA product. For example, a cell based potency assay wouldn't necessarily be used for a plasma DNA starting material, but would be used for drug substance or drug product. However, the way the RNA is delivered to the cell would be quite different for the drug substance versus drug product. This is where you would really need two separate methods or at least two separate starting points for your method. We're going to look at three different methods and three different approaches that we would take to having them be available for an organization to use to test MRNA's first, I'm going to focus on identity testing and I'm really going to zoom in more on next generation sequencing for the identity testing. But initially, what is ID testing, why do you perform it and what are you looking for? Sequence identity confirmation is a regulatory expectation in order to confirm that the product is composed of the expected sequence. In the context of mRNA, vaccines, and therapeutics, this ensures that the product contains the intended sequence, which is subsequently expressed in the target cell. Not having the correct sequence could have unintended consequences, which is why that identity test is required. Common approaches to identity testing include qPCR. Sanger sequencing and NGS. I'm going to pause here because we have a poll question on identity testing approaches. What approach do you prefer for assessing the ID of your mRNA? Is it qPCR, Sanger sequencing, NGS, or are you using some other type of approach? Looks like we're getting answers. Great. Thank you. Thank you very much. This is really helpful information as we always are striving to understand what approaches key experts in the field like you are taking. So we do appreciate that identity testing has previously been performed using a variety of molecular based methods. For example, qPCR and restriction enzyme analysis are historical approaches. However, these methods do not confirm that the entire sequence is correct, only that a portion of the sequence is generate is generating the correct profile. That could be amplification or or specific fragments or patterns on a gel, but any subtle changes would not be detected. The next evolution and identity testing with Sanger sequencing, which strives to confirm the consensus sequence, but it does miss several types of variants which may be present. More recently, NGS raised the bar due to its ability to identify and characterize low level variants in a small in a in a sample. The previous methods wouldn't detect including Sanger sequencing since this is a regulatory expectation. Fully validated methods that are specific for the intended product must be used. And as a side note, it isn't trivial to have a fully validated NGS method. Fortunately, our team has been at the forefront of development of not only NGS by NGSID testing, but in generating methods that are validated for a specific molecule target. That's why many customers come to us to look at ID testing by NGS for all kinds of modalities, including mRNA. I'm going to walk through one such scenario and to set the scene for this case study. An organization came to us to perform NGS on their final formulated drug product and received unexpected results. OK, So what we're looking at here are typical NGS results where all of the sequence statistics are provided. Key parameters which I have circled here are total number of reads mapped and the percent population mapped and percent coverage and similarity. The results show some variation when compared to a reference sequence. Similarity is only showing up at 99.78% which is close to 100% but it is not identical and this equates to A-17 base pair difference in the expected sequence versus the observed sequence. And at this point we have some questions. What are the differences in the sequence? What is the cause of this result? And perhaps most importantly, does this impact the product? Let's look at the NGS results down to the individual variance detected the NGS results give. Give you granular information about each of the variants, including the number of reads that support that variant, as well as the variant type and position. And in this example you see insertions, substitutions and deletions all in close proximity together to to each other. And I think even more importantly, they are all found, yes, in the open reading frame. This begs more questions, where did the variants come from? Is it, is it production process derived, is it raw material related with the granular information that we can obtain using NGS? We can now start to look into things a bit more to understand the potential impact to the product and how to address it moving forward. As part of the investigation, we performed NGS on the plasma DNA and the mRNA drug substance that was used prior to formulation. Previously, other approaches had been used to confirm the identity of the plasma DNA and the and the drug substance. On the bottom of the slide there's a visualization from those previous NGS results printed presented here, kind of in a more user friendly format. The purple squares marked the UTR's and the plasmid and the mRNA as you can see. Sequencing of the plasmid during the investigation showed the same sequence variants were present in the plasmid DNA as we saw in the MRMRNA drug product. This illustrates the importance of establishing the sequence early on in the process using the most sensitive methods. Had the plasmid been sequenced with NGS prior to the manufacturing run, the variance would have been detected early on and the plasmid would likely not have been used for manufacturing. It's also recommended to use the same technology on both the raw material and the final product. It's important that the testing method has the same level of detectability for variants of different types. Identification of low level variants and small insertions and deletions are not able to be detected with traditional methodology, for example Sanger sequencing. This is effectively an insurance policy. NGS is not required for the plasmid, but. Would you rather find out before you manufacture your product or after your manufacturing run that you've had an issue? Risk mitigation is a very important thing here, and the case presented happens more often than you might think. One of the key points in product testing in general is to perform appropriate testing as strategic points in the manufacturing process. As we just talked about, checking out multiple points ensures that quality materials go into the process. Checks are performed throughout and quality products come out. This helps to ensure that there are no surprises. And if there are surprises, it helps you and your investigation of the process. One of the key places to test identity is your starting material. After all, the mRNA sequence is defined by your plasmid, so a very important identity testing point in the process is your plasmid starting material. Most people sequence starting plasmids with Sanger, and this is typically what's provided by plasmid manufacturers, but that does not give you all the details. Sanger will not detect low level variants which could potentially make it into your final product. NGS will tell you if your plasma differs from what you expect. This ensures that the highest level of QC is performed on your starting material before it is introduced into the manufacturing process, and will help catch any plasmids which may contain unwanted variants. In the case presented, if the sponsor had originally used NGS on plasmids, they would have known much earlier that variants were indeed present. It is strongly recommended that you perform NGS testing on plasmid DN, AM, RNA, and final DP. The workflow looks remarkably similar for each of the steps in the process. In general, samples are extracted if required. The library preparation is performed and subsequently sequenced. During the library preparation, adapters are added to the ends of each molecule during the tagmentation process. This way, every molecule now has a known sequence on the ends, which means that the unknown portion of the molecule in the middle can be sequenced from both sides for the purposes of ID testing. Bioinformatics analysis takes all of the reads from the sequencing run and maps those reads against the reference sequence. For this analysis, a validated proprietary algorithm is used. It is the use of this algorithm as well as the expertise of our scientists that interpret and add context to the data. Insertions, deletions, substitutions and multinucleotide variants are reported. While the algorithm can identify low level variants, statistical significance of the detected variant is reported as well. Since Q 20 scores are used for the alignment. Variance greater than 1% are reported. Well, this is the recommendation for low level variant detection. This parameter is configurable to meet specific clients needs. One important thing that I want to know before I pass this over to Martin is the importance of working with a service provider that offers complete data analysis and interpretation. There are many levels of service out there when it comes to NGS having trusted GMP services. That include fully validated workflows performed in accordance with ICH guidelines is unique. Further, having validations available to regulatory reviewers through biological master files and having your having your service provider audited routinely by regulatory agencies is key. It is also critical that you work with an organization that provides more than just raw data. I don't know many people that relish having 40 million sequencing reads dropped into their e-mail inbox with no corresponding context. The product of the test is a certificate of analysis. In addition, clients are provided with download links to all of that raw data. For those folks that do love sifting through all that sequencing data, remember NGS for risk mitigation can help you avoid surprises. And with that, I'm delighted to hand this over to Martin. Thanks very much, Amy So. It's nicely described there the value of NGS testing for identity testing. I'd like to move on now and describe another technique that is really powerful for the analysis of M RNA, and that's liquid chromatography in combination with mass spectrometry. Here's a remainder of the structure of an M RNA molecule. In the center we have the coding sequence for the gene of interest, and that's flanked on either side by the untranslated region. Now at the five prime end, there should be the five prime cap. The capping consists of the addition of methylated guanosine residue to that five prime end at the other end of the molecule at the three prime end. There is a stretch of repeating adenosine units and that polyetail conveyed in length from about 20 to around 200 or so bases in length. Both the five prime cap and polyetail play important roles in protecting the mRNA from degradation as well as ensuring efficient translation. And for that reason, they are both considered critical quality attributes. We're going to move on and look at how Lcms can be used to address both of these Cqs. To measure capping efficiency, we have to be able to resolve the capped and uncapped structures. That's the difference of a single base in an M RNA sequence that is. Thousands of nucleotides long. Now such a difference is simply too small to measure by Lcms, and we can't distinguish between the capped and uncapped structures on the intact mRNA. So we have to simplify things by digesting the mRNA into smaller fragments that are easier to analyze. We do that by using a sequence specific probe that binds to the five prime end of the molecule. We then use the enzyme Rna's H, which recognizes this RNA DNA hybrid and cleaves the M RNA into a smaller oligo. The RNA can then be purified and analyzed by HPLC either with or without the Ms. detection. Now the capped and uncapped oligos should be resolved chromatographically, so we can simply measure the capping efficiency by measuring the relative peaked areas of the peaks that correspond to the capped and uncapped structures. Now in reality the chromatogram is much more complex than the simple schematic shown here. So we use the Ms. to really help us identify which are the corresponding peaks of interest. Once we've done that initial characterization, then we can simply use HPLC with UV for routine analysis thereafter. Now to assist the Poly tail. It works in a similar principle in that rather than looking at the intact M RNA, we digest that into smaller fragments. However, this time instead of using an RNH H probe, we are using the enzyme Rna's T1. This gives us lots of fragments and however we can use affinity purification to collect. The region that contains that Poly A at the three prime end. Once we have those all that goes then we can analyze them by Lcms. Now compared to the five prime end, the three prime end of the molecule is much more heterogeneous in structure and there are population of different species containing different lengths of the Poly A tail. All the goals that contain a short stretch of an Anisenes are resolved chromatographically, whereas all the goals that contain much longer stretches of polyele tend to Coalute as one broad peak. And it's in those instances that having the AMS is really useful because that allows us to resolve those different species. And allows us to determine the distribution of Poly A that's present in the sample. So LCMS is a really powerful tool for the analysis of mRNA and so it's commonly used in characterization historically high resolution Lcms systems. Have been challenging to deploy in GMP environments because of the complexity of the instrumentation and gaps in the data integrity. However, nowadays with improvements in instrument design and software compliance, it is possible to use high resolution Lcms in the QC lab. In our lab, we've successfully validated a time of flight our spectrometry system. That we're using for routine GMP lot release and stability testing. So far, Amy and I have focused on characterization and analysis of the mRNA itself, but equally important to the quality of the product is the formulation or the delivery vehicle and that's commonly. Lipid nanoparticle. An LMP usually consists of four different lipids, and each of these plays an important role. Of particular importance is the cationic or ionizable lipid, and that's required for encapsulation of the RNA as well as release of the API into the cytoplasm. Other lipids include cholesterol. Pig lipids and helper lipids, and these all contribute to the overall stability of the LMP. Now the identity and ratio of the individual lipids present has a big impact on the activity, the biodistribution and the potential toxicity of the LMP. So a small difference in the lipid composition can have a big difference. In the overall safety and efficacy of the product. In addition, the performance of the formulation and the consistency of the manufacturing process is affected by the purity of the raw materials used. And so hopefully it's clear that lipid composition is very important to the overall quality of Mr. and a products and it's something that should be assessed. Having said that, lipids are challenging molecules to analyze. Most lipids have pretty low UV activity and that limits the utility of standard HPLC detectors. We can overcome this by using an alternative detector such as charged aerosol detection. And that doesn't rely on the molecule possessing a chromophore to give a response. Now, when working with different Lmps, some amount of method development is to be expected because of the sheer diversity of lipid structures present. First, we have to consider the organic solvent that we use to dissolve the LMP into its constituent lipids, and it's important. You know that's important because it's we really must ensure that we have quantitative recovery of each individual lipid. Then from a chromatography standpoint, we need to choose a combination of stationary and mobile phases that will give the required retention and selectivity. Lipids are hydrophobic compounds and so they are well suited to reverse based chromatography, although other separation mechanisms are possible. Charged datasol detection relies on evaporation of the column element and so volatile mobile phases are required. Here's an example of some data of an LMP analyzed. By reverse based chromatography in combination with charged Datasault detection and on the left you can see a chromatogram that shows resolution of the LMP into its four constituent lipids. Each lipid is identified based on its retention time. We can then quantify the amount of each lipid present by. Looking at the peak area relative to a standard curve for that particular lipid, we also get an indication of the purity of the formulation by the clean baseline and the absence of other peaks in chromatogram. So from this one method we're able to assess the identity, quantity and purity of the different lipids present in the LMP formulation. So to summarize then, a broad range of methods is required to properly assess the critical quality attributes of mRNA products, and that involves testing both the mRNA itself as well as the formulated LMP where possible. We can reduce the time needed for method establishment by employing a combination of genetic methods and platform approaches. However, sometimes product specific methods will be required and in those instances we look to leverage our experience in working with similar products and through developing methods with standards. Amy and I have touched on the. Increased detail the powerful techniques like NGS and LCMS can provide and really that gives us a greater understanding of both the mRNA product and the process. Now it's great having access to modern technology, but that really has to be backed up by a quality system that ensures data integrity. Obviously, if you're going to be using these methods for product release, then those methods must be fully validated for GMP testing. That's something with which we've got a lot of experience and we can provide you with technical and regulatory support that you need to help navigate to the clinical development pathway. With that, we've reached the end of today's presentation. Thank you very much for joining us. Amy and I would be happy to answer any questions that you have. And so with that, I'll pass back to Mackenzie who's going to lead us through the Q&A. Back to you, Mackenzie, wonderful. Thank you so much, Martin. Thank you, Amy, for this great presentation. As you mentioned, we're going to jump into some Q&A now. Just like to remind the audience that it's not too late to submit your questions, so you can do that through the Q&A widget on the side. If we don't get through them all, we will certainly answer them offline. And as a last reminder, this webinar will be available on demand on the website soon and you'll get that e-mail notification. So with that, I'd like to go to our first question, the Amy. Oh good, we have you online as well. Perfect. So I actually think this one is going to be for you. If you have more than one molecule with the same UTR, can you use the same method for capping efficiently? Maybe I can take that one, McKenzie. I'm sorry, that one for you. Yeah, no problem. So it's a great question. So yes, if you have different M RNAs with the same initial sequence at the five prime end, then you could use the same probe for each molecule. So the method would be exactly the same. There shouldn't really be a need to redevelop the assay, but you would want to revalidate the methods for each new product just to ensure that the assay is performing as you expect. Thank you. Perfect. Thank you. Alright, Amy, you discussed ID testing of M RNA. What about single guide RNA? Oh yeah, actually so. So you can use NGS to look at ID of smaller RNA's with the organization. We've validated different portions of the workflow and So what we would do is use a small RNA prep kit which would be a bit of a change from the workflow for larger RNA molecules. But beyond that it's still a validated methods we would we would we have use a validated kit for that. But I actually think that there is also biophysical. Ways to look at this. So I'll let Martin also kind of chip in on looking at smaller R and A, Yeah, sure. Thanks, Amy. So I mean as well as a sequencing approach, you could use Lcms as the basis of an identity test. So in the presentation, we said that really M RNAs are too large to measure directly by Lcms, but it is possible to measure the intact mass of. Smaller oligos such as guide RNA you know with a pretty high degree of accuracy. And in that scenario you would simply measure the observed mass and compare that to the expected mass for that particular nucleotide sequence. And that would give you confirmation of identity. And I think you know the nice thing with having kind of different orthogonal approaches. Is that you could use one for characterization and one for release. So you have that you know those orthogonal approaches which give you that kind of strong data set. That's great. Thank you, Amy. Another one on sequencing, I think have you looked into long read sequencing technology for characterizing Mr. and I? Yes, yeah, that's a really, really great question. Thank you for the rest that so, so we So what I described today was our GMP approach using short read technology, we use the Lumina. I'm excited to share that we also do have long read direct sequencing approaches using Oxford Nanopore technology it so so this works by directly sequencing the RNA or or the DNA there's no need to generate A/C DNA library. And it sequences the entire length of the molecule. This is a very powerful tool for for ID. It's powerful for other NGS applications that we could discuss in other forums. I think it's become more of a hot topic. It's something that we we are very happy to be supporting with with customers. It's a great characterization tool. The long read technology is not at this point a GP approach, so it's really, really good for characters in chat. It helps us to see, to visualize things like in the case study I talked about today the 17 different base pair differences using the long read direct sequencing technology with Oxford Nanopore technology. Allows us to see how they are in relation to each other. Are they all within the same molecule? Are they sprinkled between multiple molecules? But even more interestingly, we can also use that laundry technology to look at things like base modification. So it's a great, it's a great tool. It's it's I think part of the future of sequencing for sure. You can tell I like this topic because I can go on and on about it. Okay. So we'll jump back to you. Apart from pad, are there any other approaches that can be used for lipid composition? So yes, absolutely. The issue here really is the low UV activity of many lipids. So evaporative light scattering detection or ELSD is a technique that is in some ways you know quite similar to CAD and that can also be used for the HPLC analysis of lipids. We prefer the Cads and just really because it's more sensitive and has a wider dynamic range. Another detection technique that you might want to consider would be mass spectrometry. So really you have. Various different analytical detectors that they could be used, but none of them really rely on, you know, UV detection or the molecule possessing a chromophore. OK, right. Thank you. And I think we have time for really just one more question and then we will close out here. Can you talk a little bit about the UHPLC based analytical methods for purity integrity for double stranded and five prime RNA capping method? That's a big question. Yeah, there's a lot there. Maybe maybe I can take that one. OK, so five prime capping. So we we touched on that in the presentation, right, But we really focused on the Ms. side of things. In terms of the chromatography you would use, I'm pairing reverse face chromatography to look at you know both of those techniques in fact the five prime capping and and polytail in terms of purity and integrity actually our go to technique for this would be capillary gel electrophoresis. But you could use chromatography, or you could use chromatographic approaches as well. So again I'm paving reverse phase. HPLC could be used to look at purity and integrity. You may also want to consider something like size exclusion chromatography to look for the presence of aggregates. Then I think the last one was double stranded RNA and so that's a good question. I mean I think it's wavely acknowledged that the analysis of double stranded RNA is particularly challenging. Our standard approach to that would be Eliza, but you know whilst we continue to offer that routinely. Our R&D group is looking into orthogonal approaches, you know, to see if there's any improved ways we can assess that particular CQA because we know it's one that's that's particularly problematic. That's great. Thank you, Martin. Their explanation in the time that we have allowed for us here. So I just I want to thank you both again Amy and Martin for this great discussion for our audience. I mean clearly you can tell our our team of experts they are excited about these methods, they are happy to discuss them further. So if you do need any support for your M RNA analytical development or release testing would like to discuss this with them. You know please you can use the the survey question on the side to. Indicate your interest and we will certainly follow up. And then lastly, I would like to call out the first webinar that we had in this mRNA series with our manufacturing unit, key benefits of PCR based mRNA manufacturing for clinical development. If you missed that first webinar last week, you can find it in the take action widget on your screen. So that is it from my side. Thank you again, Amy and Martin, thank you to our audience for attending. And I just wish you all a wonderful day. Thank you. Thank you. _1732194113633

   _1732194113860