June 26, 2024
Participants
Opening Remarks and Introduction
Oliver Ball: Alright welcome to those who've dialed in already. We're just going to give it another few seconds before we get started today.
All right. So I think we'll get going then. So Hello, everybody! Welcome to the 8th in the Unbridled Excellence Webinar Series. I'm your series host, Oliver Ball, director of business development here at Dark Horse.
We set this webinar series up to really share some of the experience, and the insights that Dark Horse has benefited from having operated now in the cell and gene space for 10 years. Over that time we've had about 380 clients in the space. So we've got quite a good amount of experience that we wanted to share with the community through this webinar series.
For those of you who've been paying attention, you'll know that AAV characterization is something that we've been talking about for quite a while now. We released the draft guidance document on full to empty ratio characterization a couple of years ago, now following the FDA's AdCom on high dose AAV toxicity which our cell and gene therapy CMC team and regs team co-authored with Nicole Pelkey.
Since then we've gone on to publish a white paper on this topic which Jacob is going to go into today, so I will not go into too much right now. But it's something that is dear to our hearts, and I think something that we feel the industry would benefit from better characterization and standards around.
Just before I hand over to Jacob, I do want to mention that our next topic, which is coming up next month will be on IND authorship. So if you're thinking about starting an IND authorship process or you've already begun, then do look out for registration on that webinar which will be opening in the next few days. Keep an eye out on our LinkedIn for registration. I think it'll be a really useful content on how to make that IND authorship process as painless as possible, to the extent that you can by means other than just asking Dark Horse to do it.
So yeah, registrations will be opening for that in the next few days. Just one other reminder is that this webinar, as all of our previous webinars are available for on demand viewing on our website. So if you want to watch the content again or share it with your colleagues, then you can find that on our website.
Alright. So without further ado, I will hand over to Jacob to introduce himself and get started on today's topic. Over to you, Jake.
Main Presentation: Advanced AAV Characterization Techniques
Jacob Staudhammer: Great thanks. Thanks, Oli. I really appreciate it, and thanks everyone for joining the presentation and the webinar today. Today we'll be going through practical considerations for implementing advanced characterization techniques into AAV release panels.
And again, my name is Jacob Staudhammer. I'm a principal here at Dark Horse. And I've been with the firm for just over 3 and a half years. Really, my primary focus is in AAV and lentiviral vector process development, process validation, manufacturing and analytics, the latter of which we'll be going into quite a bit today. And again, I've been with the firm for just over 3 and a half years.
A general ballpark has been about over 50 different organizations that I've supported and worked on in my time here at Dark Horse. So a lot of these pieces here are coming from direct experience and learnings from those projects. And additionally, previous roles that I had were at Voyager Therapeutics, Voyager Therapeutics and Encoded Therapeutics.
So today the plan is to go through this agenda here, starting with an intro and background into the space. Some challenges in implementing these advanced methods in AAV release panels. Then we'll move into a deep dive on AAV analytics and opportunities for improvement for 3 specific areas. So one is analysis of nucleic acids in AAV products using NGS. The second is empty, full and partial capsid characterization. And the 3rd is capsid protein analysis and assessment of post-translational modifications.
We'll also go through an overview of some specific regulatory guidelines for advanced AAV analytics, and some of the particular ones that should be leveraged for these advanced methods. And then we'll go into some specific conclusions and some recommendations from our experience with implementation of these advanced methods.
And I'd also like to mention that there is a Q&A button on the screen, so please feel free to send in questions during the presentation, and I'll try to monitor those as we go, and if there's anything that jumps out immediately, I'll try to address it, and if not, we can go through some questions and answers at the end.
Background and Introduction to AAV Release Methods
So, starting with a brief background and introduction about AAV release methods and advanced characterization. So one of the reasons that we've drawn a lot of focus to this is that in 2021 an FDA AdCom was held to discuss toxicity risks of AAV vectors for gene therapy products. And in short, this AdCom didn't identify a single smoking gun for why there are some toxicity risks that have been achieved with high dose AAV products. And industry sponsors, academics, and regulators are still looking for additional evidence of why some of these toxicity issues are occurring.
There are definitely some leading hypotheses, but there are still areas that are not quite clear as to why some of these toxicities are occurring. So this has highlighted a need for further advancements in the analytics world to actually characterize these factors in more depth and understand and draw correlations between any sort of toxicity, but also efficacy related pieces that we need to look into.
Additionally, AAV gene therapy sponsors have had limited guidance on the required release panels. So a lot of sponsors wait for regulatory feedback on pre-INDs and INDs before being able to set specific acceptance criteria for some of these methods. But we want to provide some better framework for folks to be able to actually set these methods and propose them themselves.
And additionally, a lot of these methods that have traditionally been used have severe limitations and methods are always being continuously developed to better characterize products. And we want to highlight some of these best practices, advice and experiences that we've had in applying these advanced methods to both clinical and commercial products.
So we want to just highlight the previous DHC white paper on AAV heterogeneity that we published in 2023. Because this kind of sets some of the groundwork for the discussion we'll have today. So this paper is really focused around the heterogeneity and product and related impurities in AAV products, and how these attributes can contribute to the product's overall quality profile.
So we split these into a few different categories. One is heterogeneity in encapsulated DNA sequence length. One is heterogeneity in DNA sequence identity and the other is heterogeneity in capsid protein integrity, stoichiometry and PTMs. And I've included a link here to this paper, and if folks want some additional reading, you can go through that link and go into this paper here.
For this slide deck presentation today, the intent is that we're not necessarily going to focus on the white paper itself, but actually look at some of the applications and advancements that you can apply to your AAV products based on some of these areas that we focus on in the white paper.
Challenges in Current AAV Release Panels
So, to set the stage, some of the common AAV release panels have really required relying on literature to set these initial release criteria. So one of those common papers that sponsors often use is what I've referenced here by Wright, which was actually published back in 2008, and a lot of these specifications and methods are still used today, although there has been a lot of advancement since then in AAV release panels and test methods. A lot of guidance to actually setting specs. Many sponsors just will rely on literature references to actually set their initial release criteria.
On top of that, though there is FDA guidance for industry on CMC information for gene therapy INDs, and that was published in 2020. But again, the specific release criteria and acceptance criteria recommendations are fairly limited.
Beyond that, including advanced methods in AAV release panels can be very challenging. This is often due to both technical challenges, so reproducibility, demonstrating that it's improved over existing methods, difficulty with individual serotypes and transgenes, so different serotypes and products can all cause challenges in many different methods. So those aren't always able to be applied to each product.
Control of the assay materials and consumables can be very difficult, as you have a very complex assay which we'll discuss in more detail later on. And then regulatory considerations, equipment compliance, and validation, 21 CFR compliance, that kind of thing. Assay validation. What kind of reference standards are you using? What kind of controls are you demonstrating linearity? Again, these are very difficult to think about from the onset of developing these methods, but something that should be considered.
And then program related challenges. So advanced assays, they can take longer and cost more to develop. This can lengthen program timelines and sponsors often want to take the shortest path to the clinic. And then, if a program already has an active IND with existing methods, changing those methods can be very difficult, because it will require bridging comparison, IND amendments. And again, that's an uphill battle that sponsors aren't always going to take.
Deep Dive 1: Next Generation Sequencing for AAV Analysis
So I want to just focus on again the 3 main areas that I discussed before and go into a deep dive for these different areas. So we'll start with analysis of sequence composition and identity in AAV products. And again, that's going to be primarily by next generation sequencing. We'll go into a deep dive on empty, full, and partial capsids. So, looking at the percentage of full, empties and partials, capsids containing either less or greater than the intended transgene, so partials and overpackaged. And then capsid protein analysis, so primary amino acid sequence, stoichiometry, post-translational modifications. And again, we'll go into some specific details on the methods that can be used for each of these, some recommendations for how to implement them, and some of our experience in actually implementing these methods in release panels.
So starting with the next gen sequencing. So the 1st area that this can be used for is analysis of transgene identity and composition. So again, typically, the identity test for AAV products is performed by Sanger sequencing. And this has been commonly used, it's proven, it's easy to operate and easy to validate. But this typically reports a single consensus sequence as compared against the reference sequence or map for alignment. But it doesn't look at minor product variants. So something that contains approximately less than 50% of the reads won't be picked up. It will report the consensus read.
And it can also have difficulty in sequencing areas with high GC content and repetitive regions, namely, ITRs in AAV products. So this can pose a lot of issues and a lot of challenges in actually using it. And next generation sequencing methods are a great alternative to Sanger sequencing for identity and composition analysis. And there's again a couple of different types of technologies within the next gen sequencing space, and I won't go into too much detail there, but depending on what you're trying to measure and how you're trying to report the value, short read and long read sequencing are a couple of different options.
In addition to the identity and composition, analysis of residual DNA is another possibility with next generation sequencing. So current FDA guidance recommends that residual DNA is kept to less than 10 nanograms per dose and approximately 200 base pairs. However, this is extremely difficult to actually measure with existing methods. FDA also requires that sponsors set limits for sequences of concern, like E1A or SV40, again, depends on your cell line and product specifically.
But there is generally across the board recognition in the challenge of reaching the 10 nanograms per dose for AAV products, and oftentimes sponsors will need to consider how to justify the higher amount of DNA in their product, and this is often done through a combination of in vitro and in vivo studies and supporting risk assessments. But this can also be very difficult, because those studies are often variable, and the risk assessments can be difficult without a lot of specific information that you can't get with methods besides next generation sequencing.
Additionally, some of the current methods use qPCR and ddPCR based assays for specific target sequences. But if you're not looking for something with those methods, you're not going to measure it. And so some of these current methods have a lot of limitations that can be solved with next generation sequencing. So again, not all residual DNA has the equivalent safety risk, and the 10 nanograms per dose, while it is in the FDA guidelines, it may not be relevant for all types of residual DNA. As a field, I think there's a lot of advancements going on to understand types of DNA that are riskier, types of DNA that are potentially less risky. And using next gen sequencing, you can identify specific sequences that are present and understand and be able to quantify their specific risks.
Commonly available commercial kits that are typically used in release panels. So things like residual 293 DNA, residual Sf9 or baculovirus DNA, those don't paint the entire picture like I mentioned. So if there's other sequences that need to be measured and controlled, using a common commercial kit potentially wouldn't measure all of the DNA that's present. So using a non-specific method like next gen sequencing will allow you to understand all of the DNA that's present. You can potentially quantitate and look at the specific sequences of concern, and this allows you to generate improved risk assessments and be able to really understand the entire profile of residual DNA that's present in your product.
So again, next gen sequencing for AAV characterization. One of the best uses of this is for analysis of the encapsulated DNA, and this can allow you to quantify partial genomes. So if you have major deletions of your transgene, or if there's impurity sequences being packaged, you can quantify the length and the amount that's present, and understand exactly what it is.
You can look at full-length transgene mutations. So if you have ITR variants, minor deletions in the ITRs, point mutations, methylation patterns. This allows you to really understand the composition and integrity of your transgene. Analysis of overpackaged sequences. So you can have chimeric reads, meaning reads that are a combination of, say, your transgene, and an impurity sequence which can occur through recombination. So analysis of these reads is important to understand that you have the appropriate sequence being packaged, and that your transgene is of the right length and size and integrity.
And then additionally, it can be used as an identity assay which I'll discuss in a little bit more detail, and really characterization of packaged sequences, and alignment to the transgene to ensure that again, your transgene is integral. Some of these outputs you can have analysis of truncations, mutations, and an overall profile of sequence lengths, integrity, and alignment to your expected sequences, and really be able to understand from a quantitative perspective, if your transgene is 100% matched to what you expect, or if there's a minor population that has deletions or mutations present.
So developing and standardizing these NGS methods for AAV analysis can be difficult. But I wanted to share some of the practical considerations that we've gone through, and would recommend for folks trying to develop these and implement them. So really, the intent of the NGS assay will influence the methods, controls and validation strategy. So are you looking at an identity assay, an impurity quantitation, impurity limits, or measurement of content? These all have different requirements for assay validation, which I'll link to the ICH guidance as well. But depending on what the goal of this method is, will have different requirements for validation and controls in the assay.
And I just wanted to note that even though you may be using the exact same equipment and general procedures, library prep methods, just a difference in the bioinformatic pipeline for these methods, so say a difference between the viral safety versus analysis for truncations, this would be a completely different assay, despite using the exact same equipment and same preparation methods. So these typically need to be kept separate. And again the assay validation strategy will be different, and the controls could be different as well.
The assay development procedures. So are you going for short read sequencing, so something like an Illumina platform? Or are you looking at long read sequencing like a PacBio or Nanopore sequencing? These are all going to be influenced by the goal of the assay. So if you're looking for long sequences where you can see different truncations or large fragments being deleted potentially, a long read sequencing would work better, whereas the short read sequencing is often going to be better for actual quantification of differences and levels of specific sequences. And this is also going to influence your assay controls. So things like spike-ins, negative controls, reference standards, etc. And the assay output. So data tables, graphs, reported values. These are all going to be different, depending on the type of assay that you're using.
And then bioinformatics strategy, data integrity. So these all need to comply with 21 CFR part 11. You need to have the specific pipeline for your data analysis within your quality management system. You don't want to have a bioinformatics pipeline that is up to the operator to decide. It needs to be well defined, version controlled, and any changes, again, need to go through your quality management system.
Setting the acceptance criteria will really depend on the type of assay that you're using and what your goal is. So are you looking for identity or impurity quantitation? That will dictate the format of the assay output and how you report the values. But really the acceptance criteria should be justified using scientific rationale, historical data and manufacturing experience. So you don't want to get to the point where you are setting an acceptance criteria without having understood exactly what that acceptance criteria means.
Finally, at Dark Horse, we really recommend using NGS to assess product characteristics as early as possible. This can be extremely useful in supporting process changes, risk assessments, assay development. And this all becomes more and more important as you move through clinical development and move into later stage development, and eventually commercialization.
There are some definite challenges with NGS assays for AAV analysis though. So strategies for assay qualification, validation are not always standardized. Many sponsors are using slightly different methods for this. And even within each assay and each system, strategies will likely need to differ.
Library prep methods, computer systems and testing labs, if you're outsourcing these especially, these are often not built with GMP and validation in mind. So oftentimes sponsors have a method with a CRO, but at a late stage, you wind up realizing that that testing lab cannot support the validation strategy that you need. And this can be extremely challenging when you find that out. So you should start with the end in mind.
Read length bias in some systems make quantitation difficult. So again, short read versus long read, that can be a consideration there depending on what your goal is. Reference standards can be challenging. So oftentimes you may not have a specific reference standard from a similar product that you can use. So it comes down to your own internal reference standards. And there's not really an industry standard for the data analysis. There's a lot of different bioinformatics pipelines out there. But at the end of the day it's up to the sponsor to define what that is.
And finally, these assays, especially if you're using an outsourced CRO, can be expensive, and the turnaround times are often very long.
Deep Dive 2: Empty, Full, and Partial Capsid Analysis
So with that I want to move into the analysis of empty, full, and partial capsids. So again, empty, full ratios are one of the most important aspects of AAV characterization. Empty and partial capsids can contribute to the overall antigenic load and impact safety and efficacy of products. So this was something that was highlighted in FDA AdComs, very specific recommendations in FDA guidance as well, and definitely something that needs to be quantified. And you should set limits prior to initiating an IND for all AAV products.
However, the field really has identified the need for empty full ratio analysis, but the field hasn't aligned on exactly what that value needs to be and how exactly to set the specifications for things like partial capsids or overpackaged capsids. This was one of the areas that Dark Horse felt needed additional guidance, and we submitted a proposed draft guidance in 2022 on specific recommendations for empty full ratios as well. And additionally, there's a significant number of groups since then that are working to standardize empty capsid acceptance limits.
So just to highlight a few common methods for analysis of empty full ratios. I'm not going to go into a lot of detail here, but I just wanted to highlight some of the common methods. So one of the main methods and typically the gold standard at this point is AUC, so analytical ultracentrifugation. So this is by far the most commonly used method. But there are other methods out there like cryoEM, cryo-TEM, etc. These are all becoming more and more popular and increasingly used.
And depending on the method, there could be more or less ability to see partials and differences in packaged sequences. So again, really start with the end in mind. Are you looking to understand the partials? Are you concerned that your product may be prone to partials? This is all something that should be considered when you're selecting your empty full analysis method.
So from our experience, we just wanted to share some recommendations for empty, full, and partial analysis. So, as I mentioned before, sponsors must quantify and set limits for empties and partials, and any non-payload containing capsids throughout clinical development. So it's not sufficient just to quantify empty and full anymore. Partials are also going to be something that needs to be understood, and with limits set.
And those limits should be set based on lots used in vivo and shown to be safe and effective. So again, this is something that needs to be defined through preclinical and clinical development, and again, will require specific analytical methods to be performed.
Again, if you're only looking at empty and full, significant subsets of product related impurities could be missed. So again, we're highlighting the need to analyze both empty, full, and partial, and that should also be paired with analysis of the packaged sequence identity as well. To really understand, if you do have partials, what the content of those capsids is and what the source is and where it's coming from, how you can control it, how you can minimize it.
And again, we recommend doing this as early as possible, specifically prior to IND enabling studies to ensure that the clinical candidate that you've selected is developed with all relevant quality attributes in mind. So it's not appropriate to wait until the middle of clinical development to start looking at these things, because again, you need to really understand them from the get-go, and that can allow you to really control the process to minimize any sort of partials.
Deep Dive 3: Capsid Protein and Post-Translational Modification Analysis
Finally, the next kind of deep dive that I wanted to go into was capsid protein and post translational modification analysis. So again, this is really an area that's new in the AAV field that folks are starting to define more clearly. But the idea is that with AAV capsids being composed of a 60 protein structure, approximately, the ratio between VP1, 2, and 3 is an extremely important consideration to both monitor and control. And this can be influenced by production methods, lot to lot variability, degradation, storage conditions, so on and so forth.
And different AAV serotypes could differ by just a few amino acids, and so slight differences in the VP sequence can lead to significant differences in the performance, safety, efficacy of the product.
Again, many current methods are limited and some current methods, some release panels rely on just a Western blot or capillary electrophoresis SDS for identification, quantification of VP protein ratios. And with certain methods like that, it may not be specific to your product, and so this can raise some concerns.
Additionally, PTMs, such as acetylation, deamination, glycosylation can occur on certain amino acid residues. But again, this will really depend on the serotype, production method, storage conditions, etc. And the impact of PTMs on safety and efficacy is currently not well understood, but it is a topic of debate in the field, and one that should be at least analyzed, considered, and monitored as sponsors go through clinical development.
So, as I briefly alluded to, the challenges with some of the current methods like Western blots: antibodies for Western blots can bind to multiple capsid serotypes. So this may not be specific to your product. And densitometric analysis of VP bands on Western blots and SDS gels is often very imprecise, and it's difficult to achieve linearity and reproducibility just from sample to sample. And so again, the CSE assays are not specific to each serotype. And additionally, these methods can't really quantify differences due to PTMs because they're not precise enough to see specific differences there.
So some of the advanced methods that can be used for this sort of analysis is LC-MS or LC-MS/MS, tandem mass spec. Additionally, native mass spec is one that can be used. And again, these are things like being able to identify the primary amino acid sequence to confirm identity, additionally quantifying and analyzing the post-translational modifications that are occurring on your capsid. And again, looking at these due to the manufacturing process and storage conditions.
So as sponsors move through clinical development, capsid identity is going to need to be performed using a method with sufficient sensitivity to really distinguish between other products in the manufacturing facility. So this becomes more of a concern if you're in a shared manufacturing facility and you don't have an assay that's sensitive enough to distinguish between other products that can be manufactured in that facility. And so again, mass spec is a great way to be able to identify your specific capsid and will be able to have enough sensitivity to distinguish between other products.
Finally, the quantitative methods for VP stoichiometry should be used, so this can be something like CE-SDS as opposed to densitometric analysis using an SDS-based gel. And then, as you move through clinical and process development, we really recommend performing characterization of post translational modifications. Again, we want to note that across the field it's not well understood which PTMs can have impacts and how those impacts can affect safety and efficacy of the product. But it is something that we feel should be measured and understood as best as possible, and we think that as the field gains maturity in this kind of analysis, there will be advancements and additional updates across the industry that could point to certain PTMs being more or less impactful, and the need to potentially monitor some of this.
With that, I just wanted to move on and focus on a couple of different regulatory guidelines for these advanced analytical methods and highlight some that I think are most important for actually implementing these advanced methods. So again, ICH Q2 is going to describe validation requirements based on the type of measured product attribute. So, as I mentioned before, depending on the type of method, so specific to next gen sequencing, if you're looking at this as an identity assay alone, the only highlighted point there is specificity, whereas if you're looking for an impurity quantitation, you're also going to need working range, accuracy, precision.
So again, start with that in mind, if you're developing this as an identity assay, think about specificity testing, whereas if you're developing this as a quantitative impurity assay, again, start thinking about the working range, accuracy, precision, so on and so forth from the beginning.
ICH Q5A R2 has actually included some specific requirements for next gen sequencing assays specific to viral safety evaluation. So this guideline has actually included some requirements if sponsors will use NGS assays to supplement or replace existing assays for virus detection. It highlights a few critical steps in these assays requiring close consideration. So these are things like sample pretreatment and nucleic acid enrichment, nucleic acid extraction and library prep, selection of a sequencing platform. So if you're again, for example, doing short read versus long read sequencing, and that highlights the bioinformatics methods and some requirements there, and it also really describes the validation requirements for these assays if there are going to be used for viral safety evaluation.
Next, ICH P14 will describe some analytical procedure development considerations. And we just wanted to highlight this one specific area of an analytical target profile. So again, this is similar to a quality target product profile which many folks are more familiar with. But this is really going to form the basis of development for these analytical procedures, which can be very useful for again selecting next gen sequencing platforms, bioinformatics methods, procedures for validation, and this is something that should be used and continuously monitored and updated as you move through clinical development.
Conclusions and Recommendations
So finally, I just wanted to highlight some of our summaries and recommendations from this presentation. Again, start early with the end in mind. Develop these assays, and use service providers that have a path to validation. We see time and time again, sponsors use an assay early on in clinical development that can't be validated or they use a sponsor. They use a CRO that potentially has some major challenges with actually achieving assay validation and appropriate controls. And you don't want to wait until the end to find that out. So start early thinking about that from the get go.
Sponsors should aim above the minimum bar for release testing characterization with a goal of continuous improvement. And again, there's a difference between kind of the minimum release testing panel versus a test panel that will get you through successful clinical development, successful process validation, successful commercialization. And as you have improved release panels and characterization methods, this will go a long way in solving root cause investigations, supporting process validation studies, process characterization, so on and so forth.
Additionally, NGS-based assays are becoming widely adopted in the AAV field for characterization, setting specs and improving processes and product understanding. So we highly recommend that sponsors start to adopt these methods in their early process and product development stages.
Identification and quantitation of partially filled capsids should be performed for all clinical AAV products. You'll really need to understand, both empty and full, but also, if you have intermediate species that contribute to one or the other, you'll need to quantify those, you'll need to identify what the content is and understand how to control that. And we highly recommend starting that assessment early so that you can select a clinical product that is less prone to partial capsids, more robust in terms of its ability to achieve full capsids as well.
Post translational modification analysis has not been widely adopted in the field just yet by the majority of sponsors, but we feel it's a valuable assay to develop during product and process development and can really support sponsors in assessing process development, making changes, developing process characterization and process validation methods. And again, there may be additional findings that the field starts to see that specifically highlight certain PTMs that could be more or less impactful to the product.
And we just wanted to highlight that again, while assay development, qualification and validation may be more challenging for complex assays, these advanced methods can really improve product development efficiency, lead to improved product safety and efficacy of AAV products. And at the end of the day the goal is for the field to advance and develop more safe and effective products, because again, that will expand our reach and ability to really treat more patients and more diseases and more indications.
So with that I wanted to thank you all for your time here. If there's any questions I'd be happy to discuss further and go into more detail here.
Q&A Session
Oliver Ball: Thank you, Jacob. That was incredibly comprehensive and detailed. I kept thinking of questions, and you answered them on the next slide. Well done. What one thing that I was interested in just asking a little bit about was, I'm kind of understanding a little bit of the FDA's perspective on some of these things like, what are the kind of regulatory implications or benefits of using some of these methods? Are people kind of at risk of creating more work for themselves by better characterization? It'd be interesting to hear your thoughts on that.
Jacob Staudhammer: Yeah, it's a great question, Oli. I mean, I think it kind of depends on the bucket here. So things like partially filled capsids and empty full, that's becoming more and more of a specific requirement. And I would be very surprised if sponsors were not asked to quantify both empties, fulls and partials at this point.
But things like PTM analysis, I think the agency, from what we've seen hasn't adopted a very specific stance on the need to monitor this from the get go. I think it's definitely a great characterization method that can inform a lot about the product. But I don't think the field has reached a consensus of the need to include that in an IND submission.
I think, for next generation sequencing analysis again, it really depends on what your intent of that assay is. If you're using that to replace something like a viral safety evaluation that has orthogonal methods, you'll need to show why that method is at least equivalent to the existing methods. And really, as you use NGS for different aspects of product characterization, I think it will kind of depend on what it's used for. But the agency, from what we've seen so far has had absolutely no objections to sponsors using NGS for advanced characterization.
Oliver Ball: Yeah, just one other quick one was really to understand what are some of the common kind of challenges or mistakes that you see people making? Like when people are thinking about trying to implement some of these methods, what should they be aware of to kind of make sure that the development and qualification of these methods is efficient and effective?
Jacob Staudhammer: Yeah, I mean, it's a great question. So I think one of the main issues that we've seen is sponsors will develop a method and implement it for early stage, and then when you reach late stage and go to validate the method, there are certain criteria that can't be met, because, for example, you can't reach the intermediate precision that you need to meet for that assay, and that's something that can easily be addressed in early development. But it's difficult for sponsors to want to spend the time and the money early on to actually develop the assay to a point where it can be validated, and that cost and time upfront ends up coming back in the end, and it often costs more time and more money to go back and develop the assays further to the point that they can be validated.
Closing Remarks
Oliver Ball: Excellent. Thank you. Well, I think that brings us to the end. I just wanted to mention, before we wrap up again, a reminder that the next webinar in the series will be on IND authorship, next month, so look out for registration on that and on our LinkedIn page, and again, a reminder that you can view this webinar and all of our previous webinars on our website. So do check those out. There's a lot of different content on there, which I think you will find interesting. So thank you all very much for joining today, and we look forward to seeing you at the next one.
Jacob Staudhammer: Thank you.