On and welcome. My name is Laurian Gute, and I will serve as your moderator today. Thank you for joining us for today's webinar. What's really in the box characterizing AAV? Encapsidated nucleic acid sequences. Using NGS as your moderator. It is my rule to ensure that we make the most of your time with us. And I'm here today with Bradley Hasson. He's the Director of Lab Operations for Next Generation Sequencing Services. Brad is a team of scientists. Laboratory technicians and bioinformaticians that perform NGS based biosafety testing services in support of product characterization and release worldwide. He has 15 years of experience developing and implementing molecular based methods for the purposes of blood release and characterization testing in a variety of biologically direct products. I'm really excited for this presentation, but before I get started I would like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets you can use. 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The webinar is being streamed through your computer so there is no dial in number and for the best audio quality please make sure your computer speakers or headsets are turned on and the volume is up so you can hear the presenter. And lastly an on demand version of the webinar will be available after. And can you access using the same link that was sent to you earlier? All right, so that's it from my side, Brad, I'll turn it over to you now. All right. Thank you, Laurie Ann, and thank you all for joining us today. Really appreciate your, your time. So like Laurie Ann mentioned, my name is Brad. I'm the Director of Lab Operations for our next generation Sequencing services here. And today I'm going to be going over how to use next generation sequencing. To characterize your encapsulated nucleic acids in your A AV production. So often times in the industry we we talk about critical quality attributes and and what those are for a particular product. Today's presentation is going to focus on identity and purity identity of your encapsulated encapsulated a AV product. As well as purity of that product as well. And 1st I'm going to start out with just a quick overview of AAV characterization of encapsulated sequences and go over next generation sequencing and how we use that to characterize the sequences. So if you look at AAV, a quick review of the manufacturing process typically uses 3 or 4 plasmid plasmids to. Produce the the A AV and it lends itself to sequencing, packaging, efficiency issues. So when you create your A AV incapsidated products, you encapsidate your expected sequence, but you also encapsidate non targeted sequences, and those non targeted sequences can consist of anything else that is in the cell based system. So it can be host cell nucleic acids, it can be plasma DNA or anything else that's present. And non targeted nucleic acid sequences can make up 1 to 2% or even greater of the total particle payload. And characterizing these sequences is important for Product Safety, so it gives you a better idea of what you're actually encapsulating and what those particles contain. And so how do we accomplish this? And so the method that I'm going to review goes over how next generation sequencing. Can be used to characterize and enumerate non targeted sequences packaged into AAV particles. So before we jump into the next generation sequencing method for this, I've got a quick whole question for you guys. The question is how do you currently characterize your encapsulated sequences during your AAV manufacturing process? We've got a couple of answers there. We've got PCR, NGS restriction, enzyme analysis. Other or if you don't currently characterize your encapsulated sequences, there's a box there as well. It is multi select, so if you do use multiple ways to characterize your sequences, please feel free to check multiple boxes. I'll give a few seconds, maybe about 10 seconds to get some answers to the questions. All right, a couple more and all right, it looks like we've got some great responses here. I'm going to Click to the next. Page. All right. We've got some interesting responses here. This is good. Thank you all for answering that question. And now what we'll do is we'll go over the next generation sequencing methodology a little bit more. But first, let's start with an overview of next generation sequencing. So next generation sequencing or NGS as I'm going to refer to in this conversation. Generates a sequence profile for all sequences present in a test sample. It is an unbiased method, and it does generate the sequences that are present in the relative quantities that they are present in. So it pretty much generates a profile of everything that's there. And then the data can be analyzed in a wide variety of ways. So a couple of different ways you can analyze the data can be for identity testing and variant characterization. For example, if you're looking to characterize your a AV based product, you can map leads to your reference sequence and get information about insertions, deletions, single nucleotide polymorphisms, or any of that nature. You can also use it for adventitious agent detection. This is actually currently a hot topic with our ICH Q5A update that came out in October, the draft update and they use. Use next generation sequencing there to characterize advantageous agents and screen for virus sequences in the product. Just another application there you can also look for inserted sequence characterization. So for monoclonal antibodies, if you have a plasmid that you've inserted into your cell line, you can look for insertion site analysis and further characterize that insert. And another element that we're going to go over today is detection of residual elements or contaminants specifically that are encapsulated. It can be used on a wide variety of samples and intermediates from viruses and cells to bulk harvest to final products and it's really the bioinformatics analysis that define the application. So next in sequencing the the actual in lab process is performed very similarly, almost identically for different applications. It's what you do with that data on the back end that sort of defines your application and and your asset. So just to quickly go over the sequencing process flow we have everything starts with a nucleic acid extraction much like most molecular biology techniques that are used. Then we move in to library preparation and during the library preparation this is specific for short read technology. A DNA fragmentation is occurs and libraries are prepared. So during that process the DNA is is broken up into smaller pieces and known sequences you can see in the yellow and pink are ligated to the unknown sequences and that is how you end up sequencing your your unknown. Now you have something on the ends of each fragment that you know the sequence of and using the sequence by synthesis reaction for short the technology. You can now build the sequence of all of your your your DNA that's present in the in the test system. Once you do your DNA sequencing, you move to your data analysis. And again bioinformatics algorithms are specific to each assay and each application. So now jumping into the analysis of encapsulated residuals a little bit more, it is more of a purity type of analysis we're looking. Add residual sequences or contaminant sequences within the capsid. And so you can use the same method that we just went over. Typically we include a nuclease pretreatment up front of the sample. We put it through the next generation sequencing method just like any other next generation sequencing assay and then we move to the data analysis now in this case. During the data analysis, what we're basically going to do is we're going to say okay. Well, we know that we have host cell DNA, we know we have plasmid DNA, We know we have the A, A/B products. So those are three of the knowns that we have. And So what we'll do is we'll take all of those hits and we'll bin them appropriately. So we'll do some remapping and we'll end up with product hits. Which are going to be your ITR to ITR with your gene of interest in the middle, That's essentially your AAD targeted sequence. We'll also map to plasmic backbones. We'll map to host cell DNA. And then of course there's always going to be some other sequences and we'll go over what that means on the next slide. And with this information, you can actually begin to express your incapsidated hits. As a percentage of the total number of sequencing hits, so you can say okay, I have maybe 40 million reads and 30 million of those are related to my A A/B. Therefore 75% of my incapsidated sequences are my sequence of interest. The other sequences we're going to map those appropriately and they may be plasmid, they may be hostile hits. And what this information allows you to do is it allows you to build a profile of your encapsulated sequences. It's actually really great for characterization of your a AV and it's really great to look at during the manufacturing process. So that way you can sort of take a look and see where you are, what your percentage of encapsulated sequences are and then you can, you know, rework some of your processes accordingly. So let's see this. In action a little bit. So I'm going to run through a case study right now. We actually have some internal collaborations here at our company and we are going to be looking at the three commonly used vectors, AAV two, AAV 5 and AAV 6. And So what we did is we took these vectors or preparations of these factors and we put them through our process, the process that I just reviewed before. To get information about what the encapsulated sequences were. And so these are the results. I'm going to take a couple of minutes to run through these results. It's the composition information gained from next generation sequencing. So if you look at the figure on the left, we'll start there. This is the AAV two. So as you can see, 87% of the sequences that are encapsulated match. To the ITR to ITR region, this is your targeted virus sequence with the gene of interest in middle. Now out of those the 13% that was remaining we mapped to human residual DNA. We mapped to plasmid sequences and we also mapped to E1A region because these are made in HA293 cell line and so that cell line was made with sheared adenovirus 5. And it contains the 1st 4.3 KB of adenovirus 5. So we included that in mapping as well. So out of the the 13% remaining 12% of the sequences mapped plasmid and 1% of the sequences mapped to human DNA. We did not get any reads mapping to the E1 region and that sort of is what our A AV do look like. Now let's move to a AV5. For the A A V5 we see only 70. 8% of the reads mapping to the a AV sequence we see 19% to plasmids, 2% to human and 1% to other. So what is other? So other can be a number of things. There are other sequences that are present that did not map to the sequences that we were screening for. Of course, if you wanted to, we can further characterize these sequences. But it's important to understand that the process of next generation sequencing actually does introduce some sequences because the transpose ACS that are used are made in a biological system. So we do get some phage introduction as well. We also didn't screen for any contaminants or adventitious agents in this particular assay. So if you look at the two, AAV two and AAV 5. You might be able to say, hey, my packaging efficiency is a little bit lower in this particular case. For a A V2 we were at 87% of encapsulated sequences mapping to the vector for a AV5 or at 78%. So now let's jump over to a A V6. For a A V6, we see 92% of the sequences matching to the expected viral product. We see 5% plasmid, 1% human and 2% other. Again, no sequences mapping to the E1 region. So out of all of these we can say a A V6 looks to be the most efficient, then comes to do and then comes five. Of course this is just within the context of the actual samples that were tested, not within it the A AV's themselves, but as you can see you can start to build a profile. Of your packaging efficiency during your manufacturing process. And so now taking that a step further, we can actually begin to look at full, partial and empty capsids to see what is in those capsids. For example, empty capsids, are they actually empty or are there nucleic acids in there? So using NGS and the agnostic approach, you can begin to build a profile of what is contained within those empty capses, what's contained in the partial and what is contained in the full. And we're actually in the process of running several of these studies right now using different fractions that we have generated to kind of build that profile. So some people may be asking hey this is all with alumina based short read technology, what is the next generation of this of this assay. And so we're currently looking at incorporating long read technology into our into our assay and what is long read sequence do long read of course is going to add another dimension to the analysis because now with long read you can actually characterize. The length distribution of this graphics, you can look at your Aavs, you can look at truncated genomes and you can look at size distributions of what is actually encapsulated. So this is something that is a little bit newer in the industry, but that we're looking into for future applications and future assays. And so I gave you an overview of that next generation sequencing method today. Our company does focus on complete safety testing packages as well as characterization. We are a trusted partner for characterization and safety testing and we do have superior scientific and regulatory expertise. So if you are interested in testing or need to test your products. We also do offer some consultancy as well to work with you and your risk assessment to determine what safety testing packages and what testing procedures would be required for your particular product. And now just jumping back to next generation sequencing for a second. We do want you to select a service provider offering more than just raw data. We do provide complete end to end analysis for next generation sequencing applications that includes all of our next generation sequencing applications. So you send us a sample. We take care of the end to end processing including the data analysis. If we're running a GMP assay, you'll get a certificate of analysis. If we're running an unregulated test or characterization test, you'll get a full report. And so we do have just run through this slide, trusted GMP services. We do have validated workflows from sample receipt to final report. We also have our assay validation data which is actually part of our FDA biologics master file. If you do file with our next generation sequencing methods with the FDA, a simple letter of authorization or LOA is required from us and the reviewer can easily access the validation methodology. If you're, if you're submitting applications elsewhere in in the world, we do support that fully and we'll get our any validation packages or SOP's directly into the hands of those reviewers. We do have an industry leading footprint. So we do have laboratories in both the US as well as the UK and we are continuing to grow our capacity to support the growing demand of next generation sequencing services. We do have secure data and reliability of results. So all of our data complies with 21 CFR, Part 11 compliance as well as data integrity that's required from all the agencies around the world. We do have a dedicated bioinformatics team as well and electronic data delivery. So all of the data that we generate is essentially yours and that you'll get to download and store or look at as you see fit. We do have expert analysis and QA review, so you do have a team of scientists. I have a team of scientists looking at your data, reviewing your data and all of our GMT based accesses are also reviewed by our quality team and also support when you need it. We do have that industry leading scientific support as well as regulatory support. If you need to consult about what tests are required for your particular product, we do offer that as well. And we do have the ability to customize methods as you need it as well for characterization. That's a little bit easier than GMP, but we can help you out when you need it. And so that's all I have for today. I'd like to thank you for your attention. And Laurie Ann, I'll turn it back over to you. Thank you very much, Brad, for this great presentation. And so now it's time to answer a few questions that have come in from our audience. I would like to remind you that it is not too late to send us your questions now using the Q&A widget. We will try to get through all of them, but if we run out of time, we will respond to you individually. And as a reminder, this webinar will be available on our website soon. You will all receive an e-mail notification when it's available for viewing. All right. So Brad here is our first question. Does this assay provide information on the total amount of residual DNA? Yeah. No, that's that's a great question. So the asset that I reviewed actually goes through a relative analysis. So we're looking at encapsidated sequences and enumerating what what is there in a relative fashion. So we're looking at total number of reads and then what portion of those reads map to which regions as as part of a whole if you're interested in total amount of residual nucleic acids like quantitative. And numbers then qPCR would be a method to determine the absolute quantitation of residuals. Okay, thank you and I can see another question which is a good one. If I already treat my sample with nucleus, can we skip the nucleus pretreatment step? Yeah. So that's a great question as well. I mean we deal with, with clients, work with clients who are sending us their products at different parts in their process and sometimes they'll have already done a nuclease treatment, in which case we can skip them lease treatment. Sometimes they haven't done it yet and we do recommend doing that as as part of the process. So that way the sequences that we identify we can, we know that they're essentially encapsulated sequences. So, yeah, that's a that's a great question. Thank you. Thank you. All right. Well, I think with this we have to close it out. So thank you very much for all the questions. If we did not get to your question, please feel free to e-mail our presenter directly. If you do need any support with next generation sequencing, I would like to discuss this method further with our experts. Please use the survey question on the side of your screen to indicate your interest and we will certainly follow up. To register for future webinars or to access our Archives Webinar library, please visit our website. So I would like to thank again Brad for this great presentation and thank you to the audience for joining us. Have a great rest of your day. _1733894612839