Hi everyone and welcome. My name is Cecile and I will serve as your moderator today. Thank you for joining us for today's webinar, The Road to approval Bioclean strategies on non blocked viral Vectors. As a moderator, it is my role to ensure that we make the most of the time of us. I'm here today Doctor Catherine Martin Remington. The Catherine Martin Remington is a technical constant in our scientific and regulatory consultancy group and she joined the organization in 2010. During her many years in viral clearance, she had others publication on the viral safety of bio pharmaceutical. Kathy earned her Ms. and KG degrees in microbiology from the University of Montana. Before I turn things over to a presenters, I'd like to cover a few housekeeping items at a bonal of the screen or multiples application widget that you can use there. You can also find a reaction button indicated by the thumbs up in which that allows you to give immediate feedback on the presentations, topics, or anything that sends out. All the widgets are resizable and movable, so feel free to move them around. To get the most out of the discount space. You can expand the slide area or maximize it to full screen by clicking on the arrows at the top right corner. If you have any questions during the webinar, you can submit them through the Q&A section. We will try to answer these during the webinar, but if a more detailed answers is needed or if you've run out of time, you will be answered later via e-mail. Please note we do captures all questions. We also have the opportunity to participate in a couple of quick full questions throughout the session. I encourage you to take part in these surveys. If you're watching this webinar on demand, you can still submit full quest, full response and questions via the Q&A widget. This webinar is being streamed through the computer so there is no dial in number. For the best of the quality, please make sure your computer speakers or headsets are turned on and the volume is up so you can hear the presenters and end. The main version of this webinar will be available after and will be accessed by using the same link that was sent to you earlier. So that's it from my side. It's my pleasure to talk things over to Kathy. Thank you, Cecile. Today we're going to talk about viral clearance for non enveloped viral vectors. Recent I I'm there, there are going to be 6 topics. I'll give you a little introduction. We'll talk about the virus panel, the importance of a scale down model, how the spiking study is done. We'll take a quick look at some data and then we'll, we'll wrap things up. So ICH Q5A revision two was finalized last November and in this revision it now includes new types of products. When this ICH Q5A was first drafted, it just covered recombinant proteins, monoclonal antibodies. But in the many years since that first version came out, we've developed a lot of new technologies. So we have things like gene therapy vectors, virus like particles. We're using Baclovirus as an expression system. And so this new version covers and includes those products and it says that viral clearance studies must be done for non envelope vectors for virus like particles and back of virus expressed vectors or virus like part particles or other proteins. We have our We have our first poll question. Yes. So we can hear from you. So what stage is your A, A product at? Is it preclinical Andy or BLA? So we're going to wait a little bit until we can collect everybody's answers. Great. So now let's see the results and thank you everyone for participating. OK. For envelope viral vectors and cell therapies, we know that any inactivation step in your manufacturing process that might inactivate an envelope contaminating virus would also denature an envelope viral vector or a cell therapy. Also, because these envelope vectors and cells tend to be much larger in size than non envelope viruses, filtration is not an option. So for these types of products, ICH Q5A does suggest that you know, some kind of viral inactivation removal measure for the culture media or any supplements are recommended, especially if what you're using is a high risk biological for non envelope vectors, however, these are very resistant to inactivation procedures. They tend to be smaller, especially AAV, they're smaller in size. So you could use a a larger poor virus filter and allow the the product vector to go through the filter while retaining larger viruses. And of course chromatography steps can also contribute to clearance of non envelope viral vectors. Well, let's talk about selecting the virus panel for a non envelope viral vector. We are used to Cho Dr. products and we kind of have that mentality. And so when we think of a virus panel, so we have to kind of go back to the roots and understand how those viruses for the choke derived products were selected. Now with AAV there are a number of ways to produce the the product. We can you know use a plasmid transfection, a stable transfection. We could use a helper virus where we're now introducing another virus into the system or we can use a Baclo virus. So the panel that you use is going to be dependent on potential viral contaminants because that's what we want to reflect in our viral clearance study. And we want to show that our manufacturing process could remove any potential viral contaminant that was introduced during the production of this virus vector. So ich Q5A talks about a number of different viruses that that you would want to think about when designing a virus panel. First of all, they talk about relevant viruses. So this is a virus that is identified as a potential contaminant of the cell substrate or other reagents or materials used in the process or some virus that is likely to contaminate those materials. An example of this would be if you used fetal bovine serum in your process, bovine viral diarrhea virus would be a relevant virus to include in the panel because that is a real potential contaminant of serum. Well, sometimes that potential contaminant isn't so easy to grow in the lab or we can't grow it to really high titers to use. So we will use a very a virus that's very closely related to that potential contaminant. We call this a specific model virus. So we want to it's usually a virus in the same genus or or family and it has very similar physical and chemical properties. An example of that would be if you're using a Baclovirus system and using SF9 cells, we know that those SF 9 cells have the potential to be infected with an insect rhabdovirus. Now this insect rhabdovirus, we cannot grow to the quantities needed and it's it's not easy to manipulate in the lab. So we're going to use a specific model virus. In this case, we're going to use a mammalian rhabdovirus, vesicular stomatitis virus to represent the insect rhabdovirus. And then sometimes there are no identified potential contaminants for your process. And so you're going to use a non specific model virus and this virus is going to show that your manufacturing process is very robust and it has the potential to remove different types of viruses, different sizes, different susceptibilities to inactivation. So you may use herpes simplex virus as a non specific model virus for a large envelope DNA virus. And then finally, ICH Q5A for the first time talks about production viruses. So these are a virus that's actually used in the manufacturing process. So this could be a helper virus, herpes simplex or an adenovirus for an AAV system. It could be bacalovirus when you are using a bacalovirus expression system. So keep this in mind. And when you're choosing your your your virus panel, make sure that you think about this kind of do a risk assessment to identify potential contaminants. And and then if there are very few, you also want to include these non specific model viruses. So here are examples for A viruses that you might used for a A V clearance. So if you're using a HEC 293 cells with a herpes helper virus, you're going to want to definitely include a herpes simplex virus or closely related virus. You could use something like pseudo rabies virus, which we're familiar with because it is also a herpes virus. It's closely related to the human herpes simplex virus. So definitely include that virus as a production virus and then some non specific model viruses to show the robustness of your system. So you may want to include an envelope, another envelope virus with an RNA genome, a couple of DNA viruses, excuse me, a couple of non envelope viruses, maybe adna and an RNA non envelope virus, similar case. If you're using 293 cells and an ADNO helper, we're going to need to show clearance of that ADNO virus, but then we can also include some non specific model virus to demonstrate robustness of your process. If you are using 293 cells and a plasmid transfection system or a a stably transfected cell line, there are probably no identified viruses as a potential contaminant. In this case you're going to want to use a panel of non specific model viruses that show that you're process can remove or inactivate a variety of different types of viruses. And then finally, if you have a baccala virus system, you're definitely going to want to show the clearance of baccala virus. You're going to want to include that specific model virus for the insect, rhabdom virus, vesicular stomatitis virus, and then you'll probably include a couple of other non specific model viruses. So we're used to doing a, a clearance study to get into the clinic with chill products. And so people a, a question people ask is, you know, do I need to do the same type of clearance study for an I&D as well As for ABLA? And definitely, if your manufacturing process includes a production virus, you're going to need to, to show that for before you get into the clinic with a, a, a, a system where you don't necessarily need to, you don't have these potential contaminants. You may or may not need to do a, a clearance study before getting into the clinic. I, I my recommendation is to talk with your regulatory body before you you our need to get do a clearance study and you know, confirm their expectation. When we think about our chow derived products, we had to do a study at least with a, a model for the retroviral like particles and we usually threw in MMV as well as kind of a worst case potential contaminant. This these retroviral like particles can almost be thought of as a production virus because they're present in the bulk harvest. And so definitely before we get into the clinic we need to show clearance of those viruses. So similarly with our non envelope viral vectors, when we have a production virus present, we definitely need to show clearance of them. Again, if you're using a plasma transfection system, no animal derived products, your risk assessment shows that you know the risk of a potential virus is quite low. I would recommend discussing it with the regulators. A lot of clients like to go ahead and perform a a study before the clinic anyway, just to give them an idea of the potential clearance capacity of some of these manufacturing process steps. Remember ICH Q5A covers data that are going to need to be submitted in marketing applications and registration packages. So it it is not it doesn't cover INDS. So remember that that there's there is that that difference and we're at our next poll question exactly. So let's hear from you. Are you looking for partner for your AAD bird clearing study? Yes, you are actively seeking a partner. No, you have a partner for your VCA A/B needs or you are considering different vendors for your VCA A/B needs. So let's wait until we can collect everybody's answers. All right, so let's see the results. And I would like to thank everyone again for participating. OK, let's move on and talk about the scale down model. I don't have a lot to say about it, but I really just want to emphasize that an accurate scale down model is really essential for any clearance study. And it it it's the same for a study with a viral vector. You want to maintain all of the the relevant parameters in going from a large scale manufacturing scale down to viral clearance scale. So you're for chromatography, you want to maintain bed heights for linear flow rate contact time with the resin. The buffers and resins should be exactly the same. PHS, temperatures, conductivities, all of those should be matching concentrations, compositions. This is so important because if your scale down model isn't an accurate representation, then your viral clearance data really aren't aren't valid. So make sure that you maintain all of these parameters and use worst case conditions for for those relevant parameters, those parameters that are relevant to viral clearance. And you know, worst case is usually the rule of thumb for viral clearance. If we don't know what's worst case, go ahead and use the targeted conditions. But yeah, for those cases where you do know worst case, please use that. So you're going to want to be able to justify to the regulators that your small scale is predictive of the large scale. You may have data for that from when you're scaling up the process. Now you're just going to scale down and if there's any deviations. From the full scale process, you need to justify those and show that that your process is you know is is still valid. I, I think it's important to set acceptance criteria for your, your study that shows your small scale is, is valid. You need to do some kind of a qualification or validation to show the validity of that small scale. And you know, this is just as important at early phases as it is at preclinical phase or. So again, the viral clearance data are only valid if the the the scale that's used to generate the data, it is a valid representation. OK, once we've selected our panel, we've have an A good scale down model, then we're ready to do the spiking study. So when we do the spiking study, it is important to do a pre study. These are experiments that are done with your process intermediate to show that it doesn't it's not toxic to the indicator cells that are used for the viral clearance study or are used to assay samples. And also that your your process intermediate doesn't interfere with the ability of that spiked virus to infect those cells. So that's important. It needs to be done a number of weeks in advance of the clearance study and also you need to use every process intermediate that you plan to test for viral clearance. So that would be like a load, maybe a flow through eluates, things like that. They all need to be tested in the pre study. When we spike with virus, we're going to use a small volume of a virus spike. So we want a good quality virus stock so that we can use a small spike. We don't want to be adding a large spike that's going to dilute the process load material. We want it to be something that's not going to interfere with with the way that study is done. You're going to include all of the appropriate controls. These might be hold controls, other controls. These are to show that the clearance you get is actually from that process step and not from some other factor. When you're looking at inactivation, you want to look at kinetics. The overall inactivation is important, but how quickly that inactivation occurs is also really critical to understanding the effectiveness of that step. You're going to want to look at how variations in processing parameters are going to impact the viral clearance. This is especially important in a late phase study. We we call these robustness studies where you know you, if you have a range for a certain process parameter, maybe protein concentration or perhaps a aph or something, you're going. If you know the worst case, you could use that. If you don't, you may. You may want to look at both ends of that range to to verify that it doesn't. The viral clearance doesn't change throughout that whole operating area of the of that parameter. If you're using chromatography columns, you need to show that you can effectively sanitize them. And if you're going to be using these columns for any length of time, any number of cycles, you'll need to look at aged resins. And we need at least two independent experiments so that, you know, these studies are, are difficult. They're they're expensive. So the agencies aren't asking for a lot, but they want at least two independent experiments to show, you know, that this clearance is repeatable. I have put this one document up here in the green box. It's, it's an old document, but it's, it, it has a lot of good information on putting together a clearance study. And I, I, it's always useful for me to, to give it a read and, you know, remember the things that the agencies think are relevant. ICH Q5A also talks about, you know, designing the studies and the things that are important. And so I, I think, you know, familiarizing yourselves with, with these regulatory documents is really important. So when we are calculating clearance for a study, we are going to look at how much virus we're putting into the process. So the the starting titer and then how much virus is leftover at the end. And that difference is your clearance, your log reduction factor, if you will. So for some process steps, especially those where you don't detect any viable virus after the processing step, it's useful to do large volume testing where we're we're actually testing more of of your sample and it gives increased sensitivity to the assay and will allow us to drop that limit of detection or quantitation if you will and increase the clearance. This is especially useful if you have a step that that has a reagent that maybe toxic to the indicator cells. And so we have to maybe dilute the sample before we assay it and that increases that level of detection. And so if we do large volume testing that will bring that back down. Well, let's take a look at some data. These are for a virus like particle that was expressed in a human cell line or the top one. We have a virus like product and the bottom one is a small non envelope virus product. The top we are the green box. We're using three envelope viruses, XMULV, which is an envelope virus with an RNA genome, bovine viral diarrhea virus. This is another a smaller envelope virus and it has also has an RNA genome. But BVDV is one of the more resistant envelope viruses and so that's nice to include as a worst case virus. PRV, pseudo rabies virus. It's a herpes virus, big virus with a it's probably almost twice the size of XMULB and it has adna genome. We're we have two non envelope viruses. We have encephalomyocarditis virus, which is a big name for a little tiny virus. It's coronavirus. It is about 30 to 35 nanometers in diameter with an RNA genome non enveloped and the clients elected to choose PPV as as well. It is of course a parvovirus with adna genome and very tiny, very resistant. So in this particular process, they had an inactivation step and a chromatography step where you see a greater than or equal to sign. That means that there was no live virus detected. So for the inactivation step, no XMULV was detected. There was a probably a little bit of BVDV detected, but we were still able to show over 4 logs of clearance. And then for PRV we had greater than 5 logs. Of course we didn't evaluate the step for EMC or PPV for the chromatography step. For the envelope viruses we got about 3 logs all the way across. Non envelope viruses, we got two logs of EMC removal and four logs, a little over 4 logs of PPV. This is interesting and I think it it shows us that in a chromatography step, the virus is acting as its own molecule, its own macromolecule at you if you will in interacting with with the resin. Each virus has its own Pi and so it's going to independently interact with the the resin. The purple box below the client now dropped back to four viruses. They they did not include XMULV in this process. So enveloped RNA, envelope DNA, virus enveloped RNA, non enveloped RNA and non envelope DNA inactivation step. This step was very effective for both BVDV and pseudorabies. No infectious virus was detected. Chromatography step. Interestingly, we saw no reduction of BVDV. We saw no detectable PRV, so greater than or equal to 5.5 logs of PRV reduction, no reduction of EMC and almost two logs of PPV. Again you know another illustration that these viruses are are interacting independently with the resin. These are some viruses that were expressed non envelope virus products that were expressed in Baclo virus. So for these, we'd looked at baclovirus that we spiked in and vesicular stomatitis virus. Here we had an inactivation step, a chromatography step and a filtration step. Inactivation step was very effective for both of these enveloped viruses. No detectable virus after the inactivation step chromatography we saw 1.8 logs of baclovirus reduction and 3.8 of VSV, and we saw good removal of the virus through a large pore filter. Similarly in a second study of a a different Baclovirus express product, we had an inactivation step that was very effective. No detectable Baclovirus or VSV chromatography step was pretty good for both viruses, over four for baclovirus and just under 4 for VSV. And again we saw a good reduction across a large pore filter. So ICH Q5A mentions that for a a non envelope vector, you may not get as much clearance as you would across the manufacturing process for a recombinant protein or a monoclonal antibody because you you may not have as many purification steps and they might not be as effective as some of the ones that are used for a monoclonal antibody. So they say that you need to make your virus safety package really comprehensive. So, you know, consider treating your raw materials, especially if there's a potential for a contaminant to be present, for example, if an animal derived product is being used. And then use extensive testing for broad virus detection of your, of your, your cell lines, your, your vector banks. You may want to consider a broad, a broadly effective method like next generation sequencing, so that just to kind of beef up your whole viral safety package. How much clearance do you actually need? You may have fewer steps. So what what is what is the rule of thumb? ICH Q5A talks about chow products and that you need to have clearance of at least 4 logs more than the the retroviral like particle count in your bulk harvest. It doesn't say anything about viral vectors. I think that's because you know there are so many different ways to produce it. They have not seen as much data for viral vectors as as they have for recombinant proteins and monoclonal antibodies. So any manufacturing system, any expression system that uses a production virus, a helper virus, baccalo virus, you're going to need to show that your manufacturing process can remove in excess of the amount of virus that was put in. So the amount of virus that would be in your, your bulk harvest before any inactivation, you need to show that, you know, that's your, your starting count and you need to show that your clearance steps can remove at least that. I, I think for other products from other expression systems, I think it's very useful if you have a good inactivation step, if you can include a, a filtration step, those are usually pretty transparent to your process. Those are really great. If you have a, a molecule that's kind of finicky, you can't use an inactivation step. I think you need to, you know, look at other steps that could potentially provide clearance and then, you know, evaluate the clearance of those steps and then also look at your whole viral safety package. Well, to wrap this up, viral clearance studies are now required for non envelope viral vectors and virus like particles. So this is new. The panel of viruses that we use might be unique to each vector product because the expression system is different. So you need to consider that and decide on an appropriate panel. This is something we could certainly help you with and your clearance might not be as robust for this product as it is for a recombinant protein. So we need to look at the total viral safety package. Again, we would love to help you design A clearance study and we would, you know, it would be our pleasure to to help you come up with your virus panel and look at the most appropriate steps for your process. And with that, I'd like to thank you for your attention. Thank you, Kathy, for this great presentation. So now it's time to answer a few questions that have come in from our audience. But before we do so, I would like to remind you that it's not too late to send us your questions when using the Q&A widget. It's also applied to our on demand viewers. We'll try to get through all of them, but if we run out of time, we'll respond to you individually. As a reminder, this webinar will be available on our website soon. All participants will receive an e-mail notification when it is available for viewing. Now back to Kathy who sort of answering questions I have coming. So the first question that we got is what inactivation process is typical? Typically we will see detergent inactivation or a low pH inactivation. Those are effective steps that have been used for a long time for envelope virus. Great, now. The second one we receive is what kind of the aspect should we select? Virus type type? Virus clearance study. Again, I think we talked about this earlier. You know, in looking at your virus panel again, you want to be aware of potential contaminants in your system. Certainly if there are any animal derived products you need to think about the viruses, potential contaminants that might be associated with those and and then you know look at nonspecific model viruses. Typically you would want to choose an envelope virus with an RNA genome and an envelope virus with a DNA genome. If they're different sizes, that's better. Same thing for non envelope viral vectors. Consider also that AAV is a parvo virus and so any and and so if you include a parvo virus like MMV or PPV your clearance may not be as great as if you had looked at at a different RNA or DNA non envelope virus. OK, another question we got is what is a recommendation approach for phase one viral clearance validation evaluation on a non enveloped in vivo delivered bound vectors example AAD. Yes, AAV, this is this is a big question. There haven't been a lot of viral clearance studies go through or go past regulators. So this is still, you know, a a concern. But I would recommend, if in doubt, then to, to do a, a, a study before your Ind. But I, I really recommend, you know, contacting the regulators and getting their input. The one advantage of doing a study before your Ind is that now you're going to have information about the potential of that step to remove or inactivate viruses. You know, a lot of times you may develop a process and and say, I I think I'm going to get this much clearance across this step, but without data, you don't know for sure. And it's really not a pleasant surprise to get to a pre commercial enabling study and find out that you're not getting the clearance that you need. Thank you for answering. Another question we got is how can next generation sequencing utilized to support very clearance validation evaluation? Yeah, next generation clearance isn't really, it's not involved directly in a viral clearance study, but it's, it can be a very effective part of your overall viral safety package. So we, we, we don't use viral, we don't use next generation sequencing to, to look at, you know, for an endpoint assay or anything like that for a viral clearance study. However, if you use next generation sequencing to to screen your your your cell banks and to screen your vector bank for adventitious agents, then you know then that that will be a a big part of the safety package, especially if if your clearance isn't as much as you had expected. Is AAD the only non involved virus for which a clearance study is needed? No, if you have and it's talked about a lot because it's probably one of the most common non envelope viruses, but adenovirus can be used, which is another it's adna non envelope virus. But yeah, any, any virus that doesn't have a lipid envelope can be used or we we should do a clearance study for. OK. Is there any advantage to performing a very clearance study before an Ind or INP if it isn't required or regulators? Well, I, I kind of mentioned that in answering the previous question. Yeah. I think there is a big advantage to doing that and it is the fact that you're going to have a good understanding of clearance for that particular step before you go into your your BLA enabling study, OK. Which product would you recommend to filter AAD Victory? I don't have a recommendation for any specific type of filter, but definitely A20 nanometer filter is not going to to work because you know your product is going to be held back. But if you use a 35 nanometer, A50 nanometer, even a 70 nanometer, those will hold back larger viruses. OK. So now due to the time, we will address one last question, that is how early in the product development should one start viral clearance studies, phase one, phase two or phase three? Well, I think before, before phase one, you need to be thinking about viral clearance. You need to be looking at and while you're developing your manufacturing process, you need to think about including steps that will provide inactivation of envelope, non, excuse me, envelope virus. Think about your chromatography. You know, realize that you're going to have to have an effective sanitization step, things like that. It's never too early to think about adding viral clearance to your your process. OK, great. So I think with this answer, we do have to close out this Q&A section. So thank you very much for all the questions. If you have any additional questions, please feel free to e-mail our presenters directly. If you also need support or would like to discuss with our experts, please do the survey question on the side of your screen to indicate your interest will certainly follow up. To register for Teachers Webinar or to access or archived webinar library, please visit our website. I would like to thank Kathy for this presentation today and I also would like to thank our atheists for joining us. Have a great day all. _1731735143156

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