Hello everyone and welcome. My name is Sandra Hahn, marketing manager, APAC by Reliance contract testing services. And I will serve as your moderator today. Thank you for joining us for today's webinar on how ICH Q5A revision affect the design of virus clearance studies. Today we have invited Steven made. Our associate director of Sales Development, APEC. He's responsible for engaging with customers regarding virus currents capability, providing technical and regulatory support, Steven work and biosafety testing field for more than 23 years, seventeen of which is with parlance. Steven has worked in a variety of role, including technical sales manager for APEC, account manager for Northern Europe, Business Development for clearance services, and senior technical specialists for cell and gene therapy portfolio. Stephen graduated with a bachelor science honor in microbiology from Glasgow University in 1999. I'd like to cover a few housekeeping items we'd love to hear from you. If you have any questions for Steven at any point during the webinar, feel free to post and submit your questions to us using the chat box. We would try to answer as many questions during the Q&A session. But if you run out of time, it will be answered to you via e-mail. We do capture all questions. And on demand version of the webinar will be available after and can be accessed using the same link that was sent to you. So that's all from my side and it's my pleasure to invite Stephen. Over to you. Thank you very much, Sandra. So welcome everyone and thank you very much for your time today. So as Sandra said, I'll be talking about ICH Q5A and how the new Division Two will affect the design of virus clearance studies. So for the agenda today, we'll go through three main parts. The first part of the agenda is really just introducing some of the major updates, the major themes from the revision to and talking about how that will impact the design of virus clearance studies. After that, we will drill down into two of those areas in more detail. So the first of those is then going to be looking at continuous processes and how we can design virus clearance studies for those types of continuous processes, both chromatography and activation and also filtration. And then we'll end the talk today looking at the new Annex 7, which talks around the the requirement and the expectation now to perform viral clearance studies for gene therapy vectors such as AAV and adenovirus. So I guess just to set the scene, if we think about the the previous revision, one of the Ishq 5A which was published in 1999. This was really focused on the vital safety of biotechnology products for products derived from cell lines of human and animal origin and was talking about the requirements for market application. And there was three core principles within that document which really drove all of the different recommendations. The first aspect, which is what we would call the prevention aspect, is where you are selecting cell lines and raw materials from reputable and traceable sources so that then you can try together with testing to show that you're not introducing any viruses or you are reducing the risk of introducing viruses into the production process. The second aspect, which is the removal aspect, is the virus clearance, where you're looking at the ability of the downstream purification process to remove or to inactivate any viruses that might be present. And then the final aspect is the detection aspect and this is where you're testing the product at different stages, for example the unprocessed bulk harvest. And you're looking to to provide evidence that there is no detectable virus in those samples. And these three core principles are still very much the case today, and still very much apply in the IC HQ 5A Revision 2. However, obviously a lot has changed in the industry over the last 2025 years. And a number of those key developments have really been the impetus to to drive the regulatory update. So here on this slide, I've indicated a number of the key themes which are talked about in the Ishq 5A revision 2. And includes things like moving away from traditional cell culture based virology assays and replacing those with molecular methods such as next generation sequencing and PCR. And also the the aim within the industry, for ethical reasons, to move away from the use of animals and again to replace those with alternatives, and in many cases that is again using molecular methods. But for those aspects which impact on virus clearance, I've indicated those in bold here and so you can see the three aspects here. So the first of those is the development of new therapeutic modalities. So as I mentioned, revision one was really focused on. Products derived from cell lines of human and animal origin are really looking at protein products primarily. Over the last 20 years, we've had the development of cell therapies and gene therapies, new types of viral vaccines and also new cell expression systems. And particularly here we're thinking about the likes of SF9 derived baculovirus expression systems. So we need to think about how that impacts the viral. Lead in studies and what new aspects we need to think about. The second point is in leveraging prior knowledge. So again, over those decades since revision one was published, the industry and the regulatory authorities have learned a lot about the manufacturing, about critical process parameters, thinking about worst case conditions from the point of view of virus reduction. And also many of those companies have gone on to develop platform technologies which they've used for many different products, potentially 10s of different products. And so they've been able to use that information to to really understand the robustness of those particular steps for their types of products. So how can that knowledge within those individual companies and also within the industry at large be used in order to make the design of virus clearance studies more efficient and more cost effective? And that's something that we'll look at today. And then the last aspect that I've mentioned here is with regards to advanced manufacturing practices and in particular continuous manufacturing. So here, you know, companies have traditionally used batch approaches and batch production and now they're starting to move to continuous processing and we need to think about what changes that might have on the design of viral clearance. And as I say in the second part of today's talk, I'll look at that in more detail. So the current status is that the I HQ5A division two draft was published on the 29th of September last year and it's now at the stage for public consultation. So organizations such as ourselves, we are discussing the new draft internally. We're also talking to our customers and we're talking to the wider industry. To understand what is important to think about any questions that we have and submitting those to the ICH for clarification. And that's something that any company or any individual can do over the coming months. But the expectation is that the document will be finalized and will be adopted before the end of this year and the current expectation is in November, so. The aspects that we'll talk through today are things that you need to think about as we move through this year and certainly if it is implemented as expected before the end of this year, then to think about that for the future and how you might have to modify your viral clearance approaches. So first of all, let's consider Cho derived recombinant proteins, which is obviously one of the major parts of the industry. And one of the key themes within Revision 2 is that modified or alternative approaches can be considered for viral clearance. So one of those first aspects is that, um, traditionally. Organizations would use xenotropic murine leukemia virus as the model retrovirus, which was the model for the CHODA arrived recombinant or sorry retrovirus, like particles that are found that are naturally expressed by those cells. One of the arguments, or one of the requests that's been made within the IC H? Is that instead of looking at MLV you could look at the retrovirus like particles themselves directly. No, that has benefits in the fact that rather than looking at model virus, you are looking at the actual virus particles which are a contaminant present and if you can directly show removal of those then that is strong data as to the safety of the product. But of course virus clearance has used the principle of model viruses for decades, and the idea is that one retrovirus should have similar physical and chemical characteristics, so another retrovirus, and so This is why MLV has been used. One of the drawbacks potentially of using retrovirus like particles is that from Cho these are noninfectious, so you're not able to look at inactivation processes such as low pH or solvent detergent. You're really restricted to looking at partitioning processes such as chromatography and filtration. So what the guidance here is saying is that if you wish, you can use the chow derive retrovirus like particles directly, but it is still acceptable to use the xenotropic murine leukemia virus and there are certain applications where that will still be an absolute requirement. The second aspect is with regards to what is called case B scenario for Cho cells and this is where you're using those Cho cells and the retrovirus like particles present are non infectious. Now in revision one there was a worked example of the theoretical dose calculation, which after that calculation was completed it gave the example of a 6 log safety margin, so one particle being present in 1,000,000 doses. As a result of that example. That that six log safety margin became the rule of thumb within the industry. So companies typically would look to have a 6 log safety margin for these products. But one of the updates and revision 2 is that although that example is still present in revision to there is a clarification that for chose cells where you're not able to detect infectious retrovirus. There is no reduction in that expectation and that now a four log safety margin would be considered acceptable. So this is helping to reduce the burden on manufacturers and to to reduce the requirement for such an extensive virus clearance study there. The next aspect is with regards to evaluation of protein a resin at the end of lifetime or what's sometimes called aged resin. So there's now a very clear statement within Revision 2 to say that that evaluation of the end of lifetime resin is no longer required for protein A. And this is, this is really driven by the type of data that you see on this slide here. So. The different regulators, regulatory authorities globally, have had many hundreds of submissions of protein a data for aged resin. And what was observed was that the IG yield dropped off significantly, whereas if you look at the retrovirus like particle clearance, you can see that that was very stable and consistent even out to four or 500 cycles. So there's a lot of confidence here from that industry data to indicate that the aged resin continues to perform very well. And in a very reproducible manner compared to to the new resin. So this is one of the reasons why that requirement has been dropped. What has also been indicated is that prior knowledge may mean that you could also take a similar approach to other types of chromatography resins. However, the expectation here is that this would be based upon platform processes and based upon data that those companies have developed themselves for different types of chromatography reasons that they are working on. So it is possible. But it does require those companies to present that data and to make a scientific justification themselves. So that leads on to Annex 6, which is talking around that prior knowledge. And within Annex 6, there is discussions around how that industry knowledge and that individual company knowledge. Could offer flexibility in the way that studies are designed for aspects such as low pH, solvent, detergent and filtration. So there's a discussion around worst case conditions and how you might take that information into consideration in the design of your study. And to take one example of that for filtration there is a discussion around the potential to apply the powerful virus log reduction for other types of viruses. So historically for a market authorization or a BLA application companies would look at four different viruses to envelope to non enveloped and a range of sizes. But filtration is size exclusion method and so you can make a very strong scientific rationale that if you can remove the smallest virus, so in this case the powerful virus if that can be removed to four logs or greater. Then it's logical to assume that larger viruses would be removed, so at least the same degree, if not a greater degree. So in theory you can perform the study with powerful virus and if you were to get greater than four logs reduction, you can claim that same 4 logs reduction for MLV aprv and reopen. Here is that the powerful virus is the worst case and if you were to see breakthrough of that virus, perhaps instead of four logs you're only able to claim 2 logs and in that case you could only claim 2 logs for MLV. And that may then make it difficult to achieve the safety margin that you want to achieve. So it's a balance here of your knowledge of the process versus the potential risks. So again this is an option, but many companies we still see using the four virus approach to take a more conservative angle. So if we move on to continuous processing, one of the big challenges here is around that multicolumn approach for chromatography and how you can approach that. But as you'll see here, I've taken a number of different quotes from the revision 2 and you can see that in each case there is discussion around using a batch manufacturing approach and applying that information to a multi column approach. So first of all there is the general statement that the basic principles are the same between a batch process and a multi column process if you're basing that on the process understanding of the science behind that step and the risk based approach. And then if we take each of the key types of downstream purification steps in turn. You can see that for inactivation, there is a comment that again, a batch process can be used as long as you're addressing aspects such as the residence time and control of dynamic parameters such as the pH range, the solvent, detergent concentration, the temperature and so on. For filtration, again a batch approach can be used as long as you are considering the worst case conditions and so here this might be aspects such as using the lowest pressure and including multiple pressure interruptions in order to assess the risk of breakthrough, especially for the powerful virus. But the most complex or the most variable step is the chromatography. But even here the ichd Q5A division two does state that even for multicolumn processes, a batch process can serve as a scale down model as long as you have well justified target process conditions. And here we're thinking about things like the flow rate and the column load versus the overload. So the last aspect or the last major theme within the Division two is around these new classes of biotechnology products. And in particular here we're thinking about gene therapy products such as AAV and adenovirus and also novel expression systems such as protein subunits being expressed using the baculovirus system. And compared to your classical chow or rodent cell derived recombinant proteins, there are different expectations for these products and different considerations with regards to. What steps can be validated and also with regards to the approach that's taken with regards to the model viruses that are used and we'll talk about those in more detail at the end of today's talk. But just to to comment on a few of those aspects, so Annex 7 has been introduced and this is a completely new part to the document which is focused on genetically engineered viral vectors and viral vector derived products. So you can see that it states now that the revision 2 does apply to these vectors. And particularly to those where you can perform viral clearance without a negative impact on the product virus. And as we'll talk about later, that does mean that you can think about implementing things like inactivation and filtration methods. And there is now an expectation that the virus clearance should be performed to determine virus reduction factors for those steps in the production process of things like baculovirus and AAV. And again, one of the big considerations here is around what are the appropriate model viruses to be used in order to consider the specific risks from those types of production processes. So let's move on to poll question one, which is focused around continuous processing. Sandra. Think it's the van. OK. Umm. Let's take a look at this first poll questions. Are you planning to implement continuous manufacturing, and if so, for which steps? Affinity chromatography. Low pH or solvent detergent in activation. And ion exchange chromatography. Virus reduction. Situations. So we like to invite you to take a few seconds to do the poll. Thank you. And then we will share the result shortly. OK. Thank you, everyone. Thank you, Sandra. So if we move on to the continuous processing aspect. So traditionally companies have used batch based process and if we look here at the example of protein a capture. One of the biggest challenges here is that you have a discontinuous loading and product recovery process and this leads to process bottleneck and slows down the overall project. In addition, in order to avoid product loss through breakthrough, you're typical only using maybe 60% of the resin and many of these types of resins are very expensive and so that drives up the cost of the process and really that together with the. Issues around discontinuous process and the bottlenecks. It really leads to two challenges for manufacturers. So as a result many of the companies now are moving towards a multi column project process and in this case you have this cyclic steady state where you are loading a column and then the breakthrough is then being loaded on to the next column. And while that's happening, you can go back to to the original column in order to do the illusion, the cleaning and the regeneration. So you have this this ongoing steady state and the real benefit here is that you're able to remove that bottleneck. And also because you have continuous loading, you're able to greatly increase the Raisin utilization maybe to to 80 or 90%. So your productivity is increased. Because you remove the bottleneck and your cost effectiveness is improved. But when we think about the challenges that that represents for viral clearance studies? If we think about a batch process, we have a single concentration of product which is denoted here by the purple line and you have when we spike that with virus, you have a single concentration of virus which then is denoted here by the blue line. So you can see they are both steady throughout the course of that process. But where we have continuous processes. You can see here from the the chromatic chromatograms below that we have these, these peaks, these pulses of product concentration throughout the course of that manufacturing. And so we need to think about how those policies in product might affect the behavior of viral clearance and especially because we may see pulses of virus over the course of that time or perhaps we we see a steady level of virus being introduced. Without that process, we need to try and understand what the impact of that is and whether that might lead to reduction in the virus clearance that can be claimed. So we've seen a lot of interest in multi column approaches from companies and continuous processes and this leads to a lot of questions around what is the appropriate downscale model here and is there a necessity to have a scaled down multi column process for virus clearance and if so, does the viral clearance vendor, do they have access to those systems and how would they potentially show that that is? Valid down scale of the client's manufacturing process. So that would be very complicated to look at and potentially would involve a lengthy investigation. But as we talked about earlier. The ICH Q5A division Two makes multiple references to the fact that if you look at those process conditions and really understand them, it is possible to use a batch based approach in order to validate a multi column study. So what we were looking to do or what we look to do is to to think about the worst case conditions for that continuous process and then to design a virus clearance study using existing equipment and existing methods. So we're thinking about a risk based approach here and really having that thorough understanding of the process and the critical parameters that might impact on virus clearance. So within the company we decided to perform an investigation. Looking at an ion exchange chromatography and thinking about how those changes in protein concentration and also potentially in the virus concentration, how that might affect the virus clearance that can be obtained. So we looked at an initial Q anion exchange resin. We used a monoclonal and we used conditions that we know are typically very effective for virus reduction. So looking at pH 8.5 and six milli Siemens per centimeter. In each experiment we had 80 mils of the monoclonal and we were targeting to spike with eight logs of minute virus of mouse or MMV, the murine parvovirus. We would then take different fractions which would be titrated for virus infectivity. So in total, we performed 5 different experiments, and I'll go through each of those in turn and then we'll talk about the results and the conclusion. So the first experiment was what we termed to be the classical batch spiking. So we had our 80 mils of Mab at single concentration. We spiked in the 8 logs of MV. We loaded that onto the column, we took the different fractions and we performed titration. So you can see from the the the graph in the middle that we have the steady state of the map concentration and also steady state of the virus titer. So this is the typical expectation for a virus clearance study performed today. In the next experiment, we decided to look at what we call a virus pulse, and in this case we had 40 mils of the monoclonal at 8.3 grams per liter. We spiked in 10 mils of MV, which gave the 8 logs, and then we had a further 40 mils of the map at the same concentration. So again, what you had here was steady state of the monoclonal concentration, but during that process. You then had this pulse of the virus coming through. So we're thinking here about how does that pulse of virus potentially affect the the log reduction that we see. The next experiment was a more extreme version of this and what we decided to call virus peak rather than virus pulse. So in this case we loaded the 80 mils of monoclonal. But what we did was we took one mil of MV, so this was a more concentrated MV. So the the 8 logs were in that one mil sample and that was introduced midway through the load. So again steady state of monoclonal concentration, but then a very significant virus titer spike or peak during that process. So again really thinking about what would be the extreme case here of of really all of that virus coming through. Not one particular time at high concentration. In the fourth example, we then thought about what would be the situation if you had a peak of both the product and the virus happening at the same time. So here we had 72 mills of the mob at low concentration at three grams per litre being loaded. But then introduced midway through the Lords we had one mil of the MMV at the eight log titer being spiked into an 8 mil sample of Mab, which was at a much higher concentration, so 56 grams per liter. So this then led, as you can see in the graph, to this spike or this peak where you have both the monoclonal concentration and the virus titer peak at the same time in that process. And then the final experiment that we performed was then thinking about the situation if we had a peak of product, but that the virus coming through that process remained at a stable level throughout. So here we have the 72 mills of Mab at low concentration and introduced midway through the load we had eight mills of Mab at the much higher concentration. And then throughout that process, we had four mills of MMV going through that process. So if we look at the results for that. You can see that actually in all cases. We were seeing significant log reduction. So typically when the log reduction is considered to be significant, this is where it's greater than 4 logs and we certainly saw that in all cases. So in all examples, we were seeing very effective log reduction here. But what's interesting is that the batch process, which is the process that we use today was giving a 5.5 log reduction and typically anything that is within one log is considered to be of a similar result. So if you think about the results here, where we have the virus pulse and the peak which are 6.16 point two, and also where you have the virus map peak at the same time and also the map peak in its own, you can see that actually all of these. Results are very similar, so you could make the justification based on this data that using something like the typical batch approach would still be a reasonable approach and could be scientifically justified for the virus clearance study on this aex. And this same approach could be used to evaluate other types of fluctuating conditions. So rather than looking at the map concentration and the the virus concentration, we could look at the pH, the sole concentration or multiple peaks of mab and virus and we can think about steps with less robust clearance, maybe things like protein A or Catherine exchange, we can think again about how these fluctuations might impact. So moving on to some of the other processes that can be used in a continuous manufacturing process. One of the new pieces of manufacturing equipment that the company has developed is a coiled flow inverter and this provides an efficient residence time distribution for low pH and activation. So this looks at inline virus inactivation by that low pH. And what what we're wanting to look at here is that if you can think about static mode, which is what we would typically look at for virus inactivation and a viral clearance study, if we can compare that to the conditions in an inline virus inactivation. What's really important here is that we are looking at that steady state within the inline process. So this is the fluids. That we are. You have a mixture of molecules with all the different residents times, so we're trying to make sure that we're covering that whole process here. So you can see with the graph and the the top corner that you have that minimum residence time and the maximum residence time and what we're really trying to make sure is that we're covering that that full scope here when we think then about the static mode virus clearance study that would be performed. And what we looked at here was? Looking at the the XENOTROPIC murine leukemia virus and activation and thinking about this over 15 minutes or over 30 minutes in the chamber and looking at the impact of different pH conditions, different buffers, whether this was citrate or acetate and comparing that minimum residence time, the maximum residence time and the steady state. And what was good was that we were able to show that there was robust inactivation of the XENOTROPIC murine leukemia virus in that in line mode for all those test parameters that we looked at. So this was data that was presented by my colleague Karen Miller at the PDA Viral safety meeting in Brussels last year. So it really shows that in addition to chromatography, we can take a batch based process or approach for inactivation steps such as low pH in this example. And then the final aspect that I'll look at from a continuous process is inline spiking for filtration. And the inline spiking process can be useful for filtration steps that are connected process. So where you have the prefilter and then the actual virus reduction filter connected. But actually many cases the inline spiking is most useful when we're thinking about problematic virus filtrations in a typical batch process. So actually where you have aggregation prone products where it's difficult to decouple the prefilter and the main filter and so you really need to think about keeping those coupled. But how can you then design the virus clearance study appropriately so you can see the setup on the right hand side? Where you have the pre filter and then we have push and pull pumps and an inline mixer in order to be able to apply the virus and then look at the virus reduction filter. And this has worked very well for some of the newer types of products coming through like fusion proteins and such a bispecific antibodies, antibody fragments where we have seen an increasing trend for more aggregation prone types of products. So the next part that I'll move on to is talking about incorporating virus reduction specific steps for viral vectors. And this is where we will have the second poll question, Sandra. Thank you 7. Our next poll question. This question is on. OK. Me. Take a look for AAV developers. That's your current manufacturing process. Include virus clearance specific steps. Yes, inactivation only. Yes, virus reduction. Filtration only. Oh yes, inactivation and virus reduction. Filtration. Or no. Thank you for taking such a few minutes to actually make your selection and then we will share the results shortly. Thank you, the police ended over back to you Steven. Thank you, Sandra. Thank you everyone. Some interesting results there. So. As I mentioned, the new ICQ 5A Division Two is now. Includes this Annex 7, which talks specifically around the requirements for gene therapy vectors. And really prior to this, the only other guideline that talked about virus clearance for gene therapy vectors was from the FDA CMC for human gene therapy I and's, which was published a few years ago. And that said that in some instances robust viral clearance studies may be necessary to remove and inactivate adventitious agents. But now we have a much more, um, direct, much more specific requirement from the authorities in order to look at virus clearance for these types of gene therapy vectors. So this is outlined in Annex 7, Section 7.3, which is entitled virus clearance. And this states that the risk of contamination with adventitious virus and residues of virus used during the production such as helper viruses and protein expression vectors. Should be mitigated following the general principles of the guideline to the extent possible. And it also states that virus clearance validation should include model viruses representative of adventitious, endogenous and if possible the relevant helper virus. So it's very clear here that where possible and especially for these non enveloped virus vectors such as AAV and adenovirus, that you should be looking to introduce virus clearance steps into your manufacturing process and where you have helper viruses. For example if you're manufacturing EEV and using adno then you need to think about that and also protein expression vectors in here. The the inference is with regards to baculovirus production. So it's really important to choose appropriate model viruses that are suitable for the production system used. So really just to kind of simplify that right down, the expectation now is that for non envelope viral therapies you should be including inactivation steps such as low key or solvent detergent and you can think about especially for AAV implementing large pore filters such as a 35 nanometer filter. So this will remove larger viruses, even larger non envelope viruses but the EV. Will be obtained in the film tree for further processing. The challenge is for your larger envelope viruses such as lentivirus and retrovirus. The problem here is that those inactivation steps will destroy the lipid envelope and your filtration is not an option because these viruses are too large. But if we think here about the the model viruses, again what we need to think about is the production system. So if you're using human cells such as heck 293 for transient transfection. Then what we need to think about is using human viruses in the process, and especially if you're using adenovirus as a helper virus, then we would use human adenovirus type 2 as one of those challenge agents. Otherwise, we can still think about using murine leukemia virus as a model retrovirus. And using the likes of PRV and Rio three in a similar way that we have done for many years for children I've products. But one of the biggest changes is if you're using baculovirus system then here you have the baculovirus which will be a contaminant and needs to be removed. And so here we would include baculovirus as one of the model viruses. You also need to think that these are insect cell derived and so rather than mammalian viruses we need to think about the risk from insect viruses. So here we might recommend the use of BVV as a model flavivirus and that then is a model for things like Japanese encephalitis virus and West Nile virus which are important viruses of concern. In addition, depending on the SF9 cells that you're using. There has been publications that many of the SF9 cells that are used have contaminations with Raptor virus. And so here we can use the likes of VSV as a model rhabdovirus in order to look at the ability of the process to remove those types of viruses. So it's important for us to discuss with you to understand what is your production system, what species of cell line are you using so that we can recommend. The appropriate model viruses to employ in the virus clearance study. And you can see here that it is possible to remove and inactivate the likes of baculovirus and VSV from an EV manufacturing process. So you can see here that things like detergent and activation can be very effective and we can look at other types of chromatography. As well as the nanofiltration again to be able to to purify and obtain the EEV while removing the larger viruses such as baculovirus and VSV. So here in these examples you can see that we can achieve almost 20 logs reduction for both the baculovirus and for the Raptor virus model the VSV. So it is possible here to provide very strong evidence of viral safety. And the last comment that I'll just make as a point to consider is that for envelope viruses, as I mentioned, it's not possible to include inactivation or filtration in the downstream process. But one of the things that you can think about is the upstream process and thinking about aspects here such as gamma radiation of the media and the raw materials or using other methods such as high temperature, short time filtration. UV treatment in order to to look at reducing the the virus load that might be present before you go into the bioreactor. So here it's thinking more about an upstream virus reduction approach rather than typical downstream approach. So just to to wrap up and summarize. So the ICH Q58 division two introduces a number of significant updates and this has really been driven by the developments and new products and processes over the last couple of decades. When you're thinking about continuous manufacturing, it is really important to think about the design of those steps and to really carefully consider the worst case conditions and how that will impact the way that the virus clearance study is put together. And then finally, for viral vectors, it is now an expectation that virus reduction steps should be implemented. We're feasible and the choice of viral vectors here is really important based upon the production cell lines that you are using. So I will finish there. Thank you to, to the audience, thank you to my colleagues who've been involved in pulling together the information for this and I'm very happy now to answer any questions that you have. Thank you, Steven for the great presentations. OK, now it's the Q&A time. We have received several questions submitted from the audience. So before we start, I would still like to continue invite the team, you know the audience to continue to post your question in the chat box. Now let's take a look at this first questions. OK. At which clinical stage the virus clearance performance need to be achieved less than 10 to the power of 6 viral particle per dose? Over to you, Steven. OK. Yeah, so. The the vital clearance is a critical part. Of the safety of the product. So there is that there is the expectation that prior to any human clinical trial that the virus clearance study is performed and that you are able to show an acceptable safety margin. So this is an expectation prior to the Phase one clinical trials. But as I mentioned earlier at least for ***** arrived recombinant proteins the the safety margin now has been reduced and so you can. A4 Log safety margin rather than A6 log safety margin. So it does make things a little bit easier for companies moving forward. OK, now take a look at the second questions. Should all the purification step counts for the performance of virus clearance? Um, OK, so. In answer to that one, what I would say is that it's not necessary to study every step in the process. So there are going to be those steps which we know from past knowledge that we really don't tend to see very effective virus reduction. And so those are steps that in discussion with the manufacturers we would recommend not to study and it is possible if you have very effective steps in the process. Such as low pH solvent, detergent filtration, it is possible to be able to achieve the safety margin that you need from a smaller number of very effective steps. And actually that's preferred by the authorities rather than having lots of steps that are only maybe giving 2 logs clear and each. So this is something that we would discuss when we're designing the virus clearance study in order to make it as efficient and as cost effective as possible but. That, no, it's not a requirement to look at every step in the process. But of course, if you wanted to be very conservative and to get as much data as possible, we can certainly do that. So it does depend on some different factors. OK. Thank you. We have another interesting questions. There's two parts to these questions. It's clearance through viral inactivation necessary for establishing overall virus clearance. If the required virus clearance is obtained without. Viral inactivation, then can the overall clearance be established? OK. So what I would say to that is that in principle. It is possible to perform a virus cleadon study. And to achieve the safety margin that you would need without an inactivation step. So if you were to have a chromatography or sorry, a filtration process which is supported by a number of chromatography steps and if they were all quite effective, if they were giving 3-4 log reduction each, it may be possible to achieve the safety margin that you need without having an inactivation step. But what I would say is that we do typically. Be that most manufacturing processes will include an activation step, so things like low pH and solvent detergent are specifically included to inactivate contaminating viruses, and they give an orthogonal approach. So whereas you have your partitioning methods. The benefit is that the inactivation is an alternative approach to be able to. Reduce the the level of virus in the sample. Now we do appreciate that some products are maybe pH sensitive, but in that case you could look at including something like solvent detergent treatment instead. So yeah, I mean overall it is possible to have a process without an activation, but it's fairly rare I would say. Thank you. OK. Now the next question is you mentioned that Cho derived retrovirus like particles could be used in virus clearance studies in future. Will data from NLP spiking studies to be accepted? Yes, absolutely. So again, as I mentioned, what the Iche Q5A division two is really trying to do is to give more options, more alternatives to manufacturers. It's not saying you must do this. And what we did in the past is no longer relevant. So the reference to using xenotropic murine leukemia virus as a model for the endogenous retrovirus is still included within the Ishq 5 beta. Vision two. So it's still very much, you know, the gold standard as it was going back over the last couple of decades, but the reference to the retrovirus like particle and using that directly is a new ID or a new approach that can be considered. And we do know, I mean you know the likes of Roche, Genentech have presented at some different conferences where they've decided to take that approach. But again there are some drawbacks to that approach and the fact that you can't look at the retrovirus like particles for inactivation methods like low pH and solvent detergent because these are non infectious particles. So certainly for those types of process steps you have to use an infectious virus like the. Ground leukemia virus. So so yes, the data from urine leukemia virus spiking studies will definitely still be accepted. OK. So due to the time, Stephen, shall we just go for, let's take up one more questions from the audience. OK. May I ask if the expectation is to include in activations and filtration steps in AAV manufacture process? How long do we take to implement and validate this? So as I mentioned there at the end, the the IH Q5 division Two does now have this specific statement that as long as it does not have a negative impact on the product, so on the product virus then you should be implementing these types of viral clearance steps. And again for the likes of AAV and adenovirus those inactivation and filtration steps can be considered. So. Really know this is the the first time that there is a specific expectation that that should be included and it really then follows on from that FDA guidance from a few years ago where there was the indication that it may be a requirement. With regards to how long to take and implement that. The, the expectation is that the Ishq 5A Division two will be adopted by the end of this year. So I would say that certainly if you're thinking about making a submission next year, then there would be that expectation that you've implemented this into your process. And obviously we appreciate that putting new steps into the process, doing all of your process validation and then the virus validation and top of that will take significant time. You know, one of the things that the likes of myself and my colleagues are doing is to to have those discussions with companies who are manufacturing AV in order to highlight this point because it is something that will take some time. And so within a year's time, we would expect that the regulatory authorities would start to ask for this type of data. That's great. So thank you and thank you for you know the great presentations. So and also here like to take this opportunity to thank everyone for joining us today's webinar. We appreciate your time. We hope to see you again next time. Have a wonderful week. Goodbye. Thank you. Thank you. _1732339006386

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