Hi, everyone, and welcome. My name is Laurian Gute, and I will serve as a moderator today. Thank you for joining us for today's webinar, Purity as a priority, optimizing safety measures for our AAV therapies. As your moderator, it is my role to ensure that we make the most of your time with us. And I'm here today with Judy Sao. Judy is a senior strategic business development specialist. She joined our company as a senior scientist in 2015, supporting the development of certified reference materials, later working as an account manager at Waters Corporation before returning to our company for her current role. She earned her PhD from the University of Pittsburgh specializing in LCMS biomarker identification for aging related diseases like Alzheimer's diseases. I'm really excited for this presentation, but before we get started, I would like to cover a few housekeeping items. At the bottom of your screen are multiple application widgets you can use. All the widgets are resizable and movable, so feel free to move them around to get the most out of your desktop space. You can expand your slide area or maximize it to full screen by clicking on the arrows in the top right corner. If you have any questions in the webinar, you can submit them to the Q&A widget. We will try to answer these during the webinar, but if a more detailed answer is needed or if we run out of time, it will be answered later via e-mail. But please know we do capture all questions. You will also have the opportunity to participate in a quick poll question during the session. I really encourage you to take part if you're watching this webinar on demand. You can still submit poll responses. The webinar is being streamed through your computer so there is no dial in number. For the best audio quality. Please make sure your computer speakers or headsets are turned on and the volume is up so you can hear the presenter. And lastly, an on demand version of the webinar will be available after and can be accessed using the same link that was sent to you earlier. Alright, so that's it from my side. Judy, I'll turn it over to you. Thank you, Laura Ann and also thank you everyone for attending this webinar today. Ernst, Laura mentioned my name is Judy Zhao, I'm a senior strategic business development specialist. The topic I'm going to talk about today is purity is a priority. So essentially this about how the AV sponsors should monitor process relate relating purities in AV drugs to ensure its safety. For this webinar, I would like to start some basic background about AVAV is a promising yet challenging therapy. There are several advantages using AV or the therapy. For example, it has low genome toxicity, it has low immunogenicity. It also has high transduction rate and sustainable expression. The bottom figure here shows the number of AAV assessed in development and the commercialized AAV drug. The total number is over 400. It also shows the number of batches produced for AAV drug across the world and the number is over 1000. And also in all these numbers are expected to grow year over year. As you can see here, AV can be a life changing medicine. However, there are still some challenges when we're using AV or the therapy. For example, it has complicated and costly manufacture process. Another challenge I want to highlight here is also related to our topic today, which is the A the impurities present in AV can cause safety concerns. Some of the impurities arise from the manufacturer process. They are called process related impurities because they cause the safety concerns. So it's the sponsor's responsibility to control and monitor these impurities to mitigate this risk. So for this webinar, so we are going to talk about the regulatory expectation for this process relating impurities and the methodologies which can be used to test these impurities. Since the process relating impurities are related to the manufacturing process. So therefore, I would like to start a commonly used manufacture process by triple transaction. As we all know that there are different manufacture process used for AAV production. So before I get into details for this process, manufacture process, I would like to get everyone's input about which manufacture process is used for your AAV programs. So there are different options here. The first one is the SF9, the SF9 cells with the bacter virus system. The second one is the triple transaction. The third one is the hack 290 hack 293 cells with the 85 health providers. The last one there is others manufacture process. So it can take a couple seconds to put your information here. OK. Thank you everyone for our input. So very interesting results here. So with that, let's move on. So as mentioned earlier, so this figure shows a high level overview of the manufacture process for AAV using triple transaction. I'm not going to go through all the details about everything here, but just want to highlight some parts related to our talk today. So at the very beginning, the cell line, for example, the hack 293 cells are thought and put into the bar reactor. In the same time, the classmate and the transfector agent are also put into the bar reactor. For the classmates, you know, they carry the general interest rabble cap jeans and helper jeans, which are very critical for an AV amplification. The transfector agent are used to get classmate into cells. So after a few days into the bioreactor, the hack 293 cells are lysed using some detergent, for example polysorbate 20 to to release AB particle from the hack 293 cells. In the meantime, the host cell proteins and host cell DNA are also released. One thing a point I want to point out here is the DNA digestion step. So the endonuclease are used to digest the host cell DNA to reduce the size like the benzonase can be used as this step. The downstream process from the clarification all the way to virus filtration are used to purify AAV also remove the impurities for the chromosography step used to here commonly used semester is affinity chromosography like the AVX ligand are used to purify AV and remove impurities. So in the end, the AV drug substance and drug products are produced. As you can see here, several critical agents added to this manufacturer process to produce high, tighter, high quality AV. However, this Regent can potentially impose safety concerns. So it is the regulatory expectation that AV sponsors demonstrates the clearance of these Regents in their AV drug to mitigate this risk. So these figures show some process related impurities in the blue boxes here. Let me walk you through this briefly. The first impurity is BSA which is added in the cell culture media to keep the cells happy. The next group is the plasmas and the transfection regions. So like I mentioned earlier, the plasmas carried the GLV interest and also some critical genes for AV amplification. The trans factor agent gas approximate into cells. The next group of impurities are introduced as the harvested step. It has this cell license Regents like the polysol by 20. They are also the endonuclease like benzonase. There's also the wholesale wholesale protein. The wholesale DNA is released from the wholesalers like Hack 293. The impurity can also introduce as the downstream process like the a faint ligand. A commonly used a faint ligand like AVX can fall off from the column resin and contaminate AAV drug. So this need to be tested. The last one here is the packaging impurity which means the AAV particles that encapsulate non vector DNA like the plasma DNA or host cell DNA. These impurities do not have the intended therapeutic functions so they need to be tested. So overall this shows the commonly seen process related impurities arising from this triple transfection manufacturing process. Again, this impurity need to be controlled and monitored to ensure the safety and quality of AV drugs. So for the next few slides, I'm going to talk in more details about why and how this impurities can be tested. The first impurity I want to talk about is the packaging impurity. So this is called out in the FDA's guideline for human gene therapy which is published in January 2020. So it points out that AAV can package the plasma DNA and host the cell DNA and the presence of this impurity can actively affect the AAV safety and quality. So the sponsor need to monitor and control this impurities present in the AAV drug. So that shows why this packed impurities need to be tested. So now the question is how we can test this impurity. We use technology called the Next Generation Sequencing, or NGS for short, for this analysis. Because NGS offers a broader characterization of the sequences packaged into AV particles, we have applied this methodology to analyze the packaging packaging impurities in different commonly used to AV serotypes like AV2AV5 and AV6. In this figure, it shows how we use this method in the lab. The 1st AV sample sample is treated using some nucleus like benzonase. So this can remove the plasma DNA and host the cell DNA outside the AV particles. Then the genome inside the AV particles are extracted and goes through this NGS workflow. The Reese generated from AV are matched against the known elements like the plasma DNA, wholesale DNA and also the vector sequence. So eventually each match is count as a hit, so we can guess the hit. For product hit, plasma height holds the height and the height for other sequences. But eventually you know we want to get the percentage of the impurity sequence present in AV particles. To calculate that we use the number of haze for the impurity sequence, for example the plasma height. This number is divided by the total number of haze obtained from this experiment. So in this way we can calculate the percentage of the impurity sequence present in AV particles. So this slide shows how the final result look like. ITR means the inverted terminal repeat, So RTR to RTR means the desired vector sequence from end to end. The plasmid and plum DNA and human DNA are unidentified and quantified. We also look at the U1A and the U1B genes because they are the transforming genes present in Hack 293 cells. So that's a requirement from FDA and the others here means the sequence introduced by the enzymatic treatment or the NGS process. As you can see here, this method can determine the percentage of packaging. In different AV serotypes. One thing I want to point out here is this work was done using the short ray technology. It's a very powerful tool, but it does have some limitations. 1 limitation is that is does not tell the length of the sequence present in AV particles because everything's chopped to small pieces. Another limitation is that it does not know how to place the RTR region because the two RTR region have the same sequence. To overcome this challenge, we use technology called long range technology. It offers the information about the sequence lens and also provide a better characterization for the RTR region. We have applied this long rate technology to AV 5 S The Ont here means the long rate technology. Lumina here means the short rate technology. As you can see here, using these two different technologies, similar result about the packing impurities can be obtained. So overall, that's why and how we we should test the packing impurities For the next one. I would like to talk about the wholesale DNA. Haft 293 cells are used for AV production carries the tumor genic genes, which can cause safety concerns. So therefore, in FDA's guideline, it points out that the sponsors can mitigate this risk by reduce the size of DNA and also reduce the amount of the wholesale DNA. In addition, the whole host cell, the I mean the host host cell like hack 293 cells also carry some transforming sequences. So these there's a risk that this transforming genes in AV drug can get into patient's cell and transform patient's healthy cells into cancer cells. So to mitigate this risk, the rescue level of this transforming genes need to be tested. So now let's look at how we can get this tested. So first for the rescue, the amount, so FDA's requirement is less than 10 nanogram per dose. Q PCR method is a sensitive method which can miss a sensitivity requirement for this. And also the primary use in this assay can be designed to target the representative human gene. So it's can also offer the specificity required for this analyzers. So that's for the wholesale DNA amount. For the wholesale DNA sizing, FDA's expectation is smaller than 200 base pair. It's actually quite challenging to analyze this. So one method can be used is the capillary electrophoresis. But if the for the rooter region, because it has the double strand DNA like structure, so it can interfere with analysis and also for some AV it has a self complementary DNA structure, it can also interfere with this analysis. The other technology can be used is the long range technology. Like I mentioned earlier, we have been using this technology working with a couple of clients developing this method for the DNA sizing analysis. The results so far are very promising. So that's for DNA sizing. The last one is for transforming genes. For HACK 293 cells that you want gene need to be tested. Like I mentioned earlier, for Hack 293 T cells, both E1 gene and SV40 genes need to be tested. qPCR method targeting the specific genes can offer the desired sensitivity and specific. So that's all for wholesale DNA. Now move on to the last impurity. I want to talk covering details here, which is the wholesale protein or HCP's wholesale protein can impose significant safety concerns to patients like causing the immunogenicity. It can also potentially affect the stability and efficacy of AV drug. So that's why FDA's guideline is caused out the rescue Wholesale protein need to be tested as a process relating impurities. There are different ways to test wholesale proteins. 1 commonly used method is Eliza. It can determine the total amount of wholesale proteins which can which have the interaction with the antibody in Eliza cage. It also in a quite sensitive it can detect down to nanogram of wholesale protein present in AV drug. There are typically commercial commercially available Kate for commonly used cell lines like host like hack 293 cell line. So this make it easy to implement this asset in the lab and also serve the need for early phase. But for the AV programs at late phase, regulatory authorities may expect sponsor to use some process specific Eliza method for wholesale protein analysis. So this is based on what we have seen for monoclonal antibody therapies. So this part of this advantages for Eliza. So it also has some limitations. 1 limitation is that. It can tell the total amount of wholesale proteins present in every drug, but it cannot distinguish between different wholesale proteins. Say for example, the AV sponsors may suspect that there are there is one or some wholesale proteins are causing the stability program a stability of their AV drugs. The Eliza acid cannot identify which wholesale protein is causing the problem. The other limitation is that the quality of the data for Eliza asset is highly dependent on the quality of the antibody used in the Eliza cage. For you know whether the antibodies provide a wide range or whether the antibody have a good authentic binding to the wholesale proteins. Even though the sponsors can have a very good batch of this antibodies, but the supply of this antibody is infinite. So a new lot of antibody needs to be produced to miss the demand. So if that happens, a bridging start need to be performed to demonstrate comparability of the different lot of antibodies for this Eli's assay. So this can be expensive and also time consuming. To overcome this challenge, an orthogonal method can be used for host cell protein analysis, which is LCMS BAG. In this method, protein including wholesale proteins and the AV capsid proteins are digested using trypsin and then the resulted peptide mixture is analyzed by LCMS BAG. So this can be used to identify wholesale proteins present in weight drug above the detection limit. And also sponsors can combining with the literature and historical data to perform the risk assessment and also identify the potential problematic wholesale proteins. So this is typically done say in a non targeted fashion. So if the host problematic wholesale protein is identified, a targeted RC mass pack master can be developed to monitor this problem. Problematic proteins in the in process, in process sample or for the loss release purpose. Nothing I want to point out that is our mass pack can provide semi quantitative information. We can spike unknown amount of protein standards into the AV sample, choose semi quantified wholesale proteins in the AV samples. However, there's also challenges associated with this method because most of the proteins present in off sample is the capsid proteins. Wholesale proteins is only present at a very low level. So this make is very challenging to detect. Some wholesale proteins has very low abundance. So that's for the wholesale proteins. So before I move on, I would like to get your input about these four question which is have you experienced the any problematic wholesale proteins in your AB programs? Have you considered using LCMS pack for wholesale protein characterization? There are also four choices. The first one is yes to both, the second one is no to both. The third one is yes to the first question, which is you have experienced the problematic host of proteins but you haven't considered using LCMS pack for wholesale protein characterization. The last choice is so far you haven't experienced any problematic wholesale proteins but you are thinking about using LCMS pack for your host cell protein characterization. So you can take a few seconds, put your answer here. OK. Thank you everyone for our input. Again, this is very interesting result here. So with that so we can move on to the next few slides. Yeah, so you know the previous slides covers the three major process relating impurities. I want to cover today for some other impurities I don't have time to cover so I listed them in this table. So depending on the nature of the impurities and also the sensitivity requirement, a suitable method should be chosen to miss the needs. For example, Eliza as say is typically used to detect BSA endonuclease like benzonase and also the authentic ligand like AVX and also the qPCR assay can be used to detect the restioplasmic EAV sample for the cell license Regent like the polysorbate 20. UV spectroscopy and the HPRC methods can both be used here. But based on words we have seen so far, large amount of AAV sample is needed in the UV spectroscopy method. The volume requirement is about 5 milliliter. So this is a small volume for monoclonal antibody, but it's a huge number for AV programs. So HPRC master ticker required much less sample is around the microliter range. So it's can it's better suits the needs for AV developers. So overall, you know us a suitable method should be chosen based on the needs for the AAV programs. So that's pretty much worse I have today for process related impurities. As you can see here in the testing, these impurities require a lot of expertise and highly skilled scientist. Although it's very challenging but still manageable. We have a broad range of services and and expertise to support you from consultation to master development to master validation. So you know we are always here to collab with you, to work with you so you can de risk your AV programs. So please come talk to us. We have extensive experience in this field and highly trained scientist. We also have a great deal of regulatory and technical expertise. We can also leverage our global network of capabilities to assist you with every stage of your AAV drug development. So look forward to work with you all. So with that, that's all I have for today. Thank you very much, Judy, for this great presentation. Now it's time to answer a few questions that have come in from our audience. I would like to remind you first that it's not too late to send us your questions now using the Q&A widget. We will try to get through all of them, but if you run out of time, we will respond to you individually. And as a reminder, this webinar will be available on our website soon. You will all receive an e-mail notification when it's available for viewing. All right. Now let's go to the questions. There is one first question, which is for the testing you mentioned in the presentation. Are they off the shelf assays? You're on news, Judy. Sorry about that. Yeah, that's a good question. So it's it's depends on the assays. Some of the assays off the shelf like Eliza Assay for BSA for Benzonase and also the NGS short range technology for the packed impurities they are off the shelf assays. We also have off the shelf assay for trans factory agents like Pi. For some newer trans factory agents in the marketplace we have a generic non GXP method. We typically write 1st to make sure it's suitable for the client matrix before we perform the face appropriate validation. So overall it really depends on the impurities. Depends on the assays. We have off the shelf assay for some impurities but for some impurities. For the other impurities, we have the capabilities to develop the assays for those impurities. We don't have off the shelf assays. Hope that answered the question. Yeah. Thank you very much. Sure. We have a second question. You discussed NGS for packaging impurities. Can you use NGS for assessment of Mt full ratio or would you recommend another technique for that? Yeah. So based on my understanding, NGS is not the is not really suitable for to determine empty and full ratio for AV drug. The golden method is the analytical ultra centrifugation or AUC. However, the challenge of using AUC is is hard to run this in the GMP environment due to the software issue. Another method is an exchange chronography or a EXAEX can be used for loss release but it cannot distinguish in the particle partial particle from the empty and full AV particle. There are also some other methods like SEC, malls, mass for photometer. Each methods have their own pros and cons. I believe there are some literatures out there talking about this. The right method should be chosen based on the needs of the aid programs. Thank you. Sure. Due to time, maybe let's take one more question. What sort of HCP's would be considered promomatic? Yeah, I believe this is probably related to the specific AV programs. But I I found a paper published in I think last year by Jonathan Bones from the University of Dublin. If I remember this correctly, they in this paper it mentioned several commonly seen problematic wholesale proteins, including several heat shock proteins. It also shows how to use LCM aspect to quantify these proteins, so you can find more details in that paper. Thank you, Judy. Sure. All right. And I think with this, we do have to close out now. Thank you very much for all the questions. If we did not get to your question, please feel free to e-mail our presenter directly. And if you need any support with your AV therapies, I would like to discuss with our experts. Please use the survey question on the side of your screen to indicate your interest and we will follow up. To register for future webinars or to access our archived webinar library, please visit our website. Thank you again, Judy, for this great presentation and thank you to the audience for joining us. Have a great day. _1733912202836

  
    
  _1733912203077