Analytical Biochemistry Strategy for Gene Therapy Drug Development by Dr. Nesredin Mussa

This is a highly emerging, highly active area of drug development. There are over 15 approved gene therapy molecules in the US. My talk is going to be what are the analytical strategies required to ensure successful development and commercialization of gene therapy assets?

Welcome to Emory Pharma speaker series. Today we're honored to have Dr. Nes Musa here presenting on a very important topic, analytical strategies for gene therapy drug development. Dr. Musa spent a number of years working on his master's and PhD degree at University of Surrey in UK. He spent 11 years at Lanza in progressively expanding roles and five years at Bristol Myers Squibb and another five years at Ultragenics gene therapy cell therapy orphan disease company that's headed by my friend Emil kakis. And recently Dr. Moussa started his own consulting company, Dynamic Biologics Consulting. And we're obviously a client of Dr. Moussa and love to collaborate with you watching this video and of course Dr. Musa on your analytical project. So with this I'm going to pass on the microphone to Dr. Moussa.

Thank you Ron Hamid, thank you for your invitation. Very happy to be here. Emory Pharma, a very good company and a very good friend. Ron, this is a good occasion for us to discuss and collaborate with on advanced medicine, analytical strategy and technologies. So I think Ron, as we discussed over the years, this industry is evolving. The evolution is basically historically we used to use natural healing and small molecules. And then after many years of small molecules now we came into the advent of biologics, protein based therapeutics where we try to cure diseases at a cellular level. The latest over the last 20 years we have progressed our technology to gene therapy. So the topic today is going to be around analytical strategies for gene therapy drug development. So we'll try to mention about gene therapy drug development but our focus is going to be what are the analytical tools required to strategize, to manage early phase development all the way to commercialization. So this is me, I have my own consulting Firm, Dynamica, managing CMCs, analytical development, quality control, due diligence and selection of CEOs and CMOs as well as technical team development. So Ron, thank you for your custom. So let's talk about the topics I would like to discuss today. We have limited time is one of the first but we'll try to cover three areas in terms of gene therapy, drug development and how analytical strategy is a critical element in the drug development activities for gene therapy. So gene therapy, drug development and what are the clinical quality attributes for the drug itself and what analytical tools do we have to help us manage the development activities and commercialization of gene therapy assets. So this will be the key topics for our discussion and to discuss those effectively, I would like us to mention what is the relevance of cqa? CQA has critical quality attributes for the drug product which are in important to help us manage the manufacturing process to ensure we have consistent manufacturing process. And how do we select the methods to use to develop and commercialize gene therapy molecules. And I will give an example in the form of case studies select technologies to discuss to highlight the analytical strategy for this new advanced medicine modality. Gene therapy. So over the years, in addition to the evolution from small molecules to protein based therapeutics to gene therapy, the gene therapy landscape itself has been evolving pretty aggressively over the last six to eight years. So now in the clinic we have over 500 clinical trial projects in the US globally, if you include EU, other Asian countries, it may go up to thousand or more. In the US there are over 500. This is a highly emerging, highly active area of drug development. There are over 15 approved gene therapy molecules in the US and while that's happening in parallel to that, we also advanced our gene editing technologies. So the gene editing technology is also advancing at a rapid rate led by CRISPR and others. As you might have heard, end of last year, the first gene editing molecule was approved by MHRA in the UK to treat beta thalassemia. So these programs are advancing really fast, but they are also getting more refined and far more acute in trying to resolve disease indications. Not only treatment, but also trying to find root cause and rectify the root cause of monogenetic disease indications. So why is this relevant for us to discuss this? The motivation for this is this area is growing so, so fast. There is a lot happening, there is a lot of activities and getting involved in this area is really eminent and really beneficial. So the money spent in development and commercialization activities in 2022 was around $15 billion. Now if we go, the projected expenditure in this area is going to be around $80 billion by 2032. This projection is really dramatic. So that's the motivation. This is very active area. There is a lot of need, there is a lot of services required and support requires to make this happen. And of course the area of development are multiple. When we say advanced medicine. In gene therapy we are including cell therapy as well. So solid cancers, infectious diseases, eye disorders, blood disorders, as well as hematological malignancies and neurological disorders are included in this progressive clinical development area of genetic therapy as well as cell therapy. So as a Form of introduction My talk is going to be what are the analytical strategies required to ensure successful development and commercialization of gene therapy assets? So what are the key analytical strategies that we need to pay attention to? This applies to all gene therapies and cell therapies as well. But the criticality of having the right analytical strategy is majority of the companies developing gene therapies and cell therapies are small and medium sized biotech companies. So there are multiple limitations, timing, financial limitations that make it important to be strategic. So the money used, the effort spent in the early days will benefit in the later days of the late phase development and potential commercialization. So the process optimization activities that happens early with any drug product, this process development activities for gene therapy are very meaningful, meaning that there is high value to those process optimization activities for one reason, because there is limited experience in this area we are all learning. Therefore when we develop a gene therapy asset, we need to make sure that we have the right critical quality attributes defined for the program for the drug early on. Much earlier on it could be presumptive CQAs critical quality attributes, but the earlier we define them, the better chances we have of granulating the right analytical strategy and we will focus I will show you in my next slide. We are going to focus on recombinant aav. There are multiple platforms to deliver gene therapy. We'll focus on associated adenovirus vehicle for delivering gene therapy and for that having an early understanding of CKAs with process development activities is important to garner meaningful analytical strategy. Once we do that, once we understand the criticality of certain quality attributes, then we will be able to select the right analytical tools and phase appropriately qualify and validate them to support eventual commercialization. So as a form example, just to show you, to give you some example, these are the platforms used to internalize the gene payload into the patient and basically you have multiple options. Our discussion will be sent around AAV today. But to show you the gene editing area is also growing. There are pros and cons benefits and some disadvantage to both of them. For the AAV based gene therapy, mainly addition of the right gene to the patient population that is more established than the gene editing technology. So there is benefit in that experience. The agencies are more familiar with it as well as we know that it's going to be long lasting. We have some experience with it. The new area of genetic addition when we can silence we can delete sequence that thereby resolving the root cause of the disease. That's much newer but actually treats the core disease area but still emerging technology. So we have a lot more to learn. One of the issues, one of the potential disadvantages for the AV based gene therapy is there is safety issues. We need to be aware of genotoxicity and potential immunologic reactions. So we need to be aware of that. The immunogenicity as well as we are not suppressing the negative traits of the genetic disease, we are just adding the the right. So we don't know what that means in terms of long term, how will that behave, how will the patient behave in terms of having the diseased genetic formula as well as the rectified? So we need to wait and see what would be the outcome. So for aav, why do we choose AAV more predominantly than other delivery vehicles? Number one, AAV is less pathogenic to humans and we have more data. Once the gene is delivered, we believe it will remain, that deliver will remain. So it protracted genetic delivery and expression in the system as well as low immunogenicity relative to others. So this is some of the benefits that people prefer to use recombinant AAV to other delivery vehicles. Why do we need to strategize effectively? It's because of the evolving landscape, because the limited budget, limited timeline. Because again, we don't have protracted timeline dealing with orphan diseases, rare diseases. The available time space is limited as well as the budget aspect, also very limited. Therefore, we do the necessary work and we make sure that we are able to deliver these much needed treatments to patients as quickly as possible. Of course the agency approval is critical and the complexity of the programs, these are not simple programs, they are very complex. The complexity is because of our experience. We are still learning how to make them better, how to make them more swiftly, how to make them more reproducible, how to scale up effectively, how to improve the title. So we are still learning. Therefore, that complexity makes it more challenging to manage than traditional biologics or even definitely small molecules. So we are all growing together. The agencies are knowing more about this, we are learning more. The more we are working on, the more programs we work on, the more challenge we are facing, the greater the learning. And of course the agencies, they know more than us because they are exposed to far more applications, they have far more visibility to multiple forms, development forms and multiple approaches. So as a result, this area is still growing, the opportunities are even high, they will remain high for the foreseeable future. So basically, in terms of development, once the indication is identified and proof of concept is established by translational sciences, we go on to make sure that this process is developable, we need to make sure it is developable and eventually manufacturable. So we need to assess developability and manufacturability. Therefore, the engagement of translation sciences with development teams and operation teams in a suite team session is critical. We need to have the right scale down process models to be able to scale up, down the line, as well as to learn about the right analytics that will help us manage the program, the product quality, we need to challenge the process, the product and analytics using accelerated stability studies and forest deck studies. So we need to screen the technology as well as the conditions at the early stages to make sure we have developable and manufacturable assets. That is the process development in a nutshell, it is a big chunk of work. The main CMC activities that. But having the right CQA to support the manufacturing is very important. That consistent manufacturing will require the guidance of the right CQA is being defined that will deliver the right dose to the patients. So here we will discuss some of the relevant critical process steps and the relevant CQAs. So when it comes to CQA assessment, we need to make sure that we think in the right direction. From early on, we have to set with the end. We have to set our eyes on the end game. We begin with the end in mind. Where are we trying to go? So eventually, when we have the intention is we want to commercialize, Therefore, when we experiment early on, the target quality product profile at that early stage, when we define that we need to think what kind of technologies are required to support this. And with that, when we define that, that will eventually help us assess what kind of CQAs we need to develop for us to be able to commercialize this. So the target cost profile, which means that what are the target qualities required for this program for this product to perform as expected, as intended? And then once we do that, we need to develop the right technology, the right analytical technology, select the right analytical technology to assess the CQAs, determine the CQAs, and with that we will define the right critical process parameters, the CPPs. So, so that the process will be able to be used to manufacture on a continuous and routine basis. And of course we need to also define the critical material attributes, because what happens is that the process and the material are very critical elements to give us the right target profile and the right sequence are linked, so they feed into each other. So therefore, we need to ensure we think about this early on. And of course the mechanism of defines we have the target quality profile we identify the CQAs, then we identify the key process parameters during development stage. Then we define the upper and lower limits of the cma. The cma, what are the critical material and we define them, we validate the vendors and, and we make sure we don't change unless we qualify the new vendor. And the CPPs are important to be able to generate the right target quality profile continuously. There is a loop, early loop. This gives us a good mechanism of understanding how they all talk to each other. So we have to be in a sweet team setting, especially in a small company. It's important we interact effectively to make sure we are on the same page and the same thing here when it comes to the analytical technology that we use. Because we have to use certain analytical tools early on to develop the program, we have to be cognizant that this analytical tool may be used for release. They may also be used for in process testing. So we have to keep in mind all the technologies used during early translation, translational science stages, during early development stages. They are potential technologies that can be used for release or long term and extended characterization purposes. So we need to think about fit for purpose technologies early on because of the compressed timeline and also reduce, you know, when we are going for orphan designation that's very critical. So just as an example, this is a flow of platform manufacturing process. So without going into detail, we get, we grow the cells up to the manufacturing scale. It could be 500 liter or up to maybe 2000 liter cell culture. Then that goes through the depth filtration cell lysis to get the aav. Once we get that, we concentrate and go into the applicable chromatography steps. Then we ultrafilter and dye filter into the right formulation and then we have the sterile filtration for drug substance, eventually drug product. This process needs to be defined as well as the critical quality attributes for each step has to be defined. We have to establish a risk based assessment. What are the critical quality attributes attributes at each of the process steps? This is very important because when we get down to the downstream to alter filter to get into our drug substance, we are on the right track. So we start doing this by developing risk based CQA assessment, risk based CQA definition and identification. Again, when we are talking about risk based, we have to think about what do I need for phase zero and what do I need for phase three and commercial and eventual life cycle. So the risk based metric assessment of CQAs would look like a matrix. We need to identify which ones are more critical than others and that matrix will help us, will guide us to identify the right CQAs for each phase of the programs. At Phase 0 and at Phase 1, Phase 2, Phase 3 and Commercial, the criticality of these quality attributes will increase. But we define that much earlier. You define early on. We start with the presumptive CQAs until we get late phase CQA, then commercial CQAs. These are risk based because you don't want to boil the ocean. You don't want to make everything as a critical value attribute. It has to be the right one. So that's why we need to have a risk based approach here. So that is the basic introduction into from the process manufacturing, process development, manufacturing process. How we define the CQAs in a generic term then now how do we select the right methodology? How do we assess the right technology to actually define the right CK's and assess the right CQA? So here I divide them into three sections. So we have got. Whenever we are talking about analytical strategy, you are talking about three buckets. Bucket number one with the most important bucket is the release assays to release drug substance, drug product. The bucket number two is extended characterization, which is very important, maybe lower in a hierarchy, but just as important. And the third bucket is in process testing. They are all critical for an amicable drug product development. So having the right technique at each of these stages for each of these buckets will facilitate early submission, will facilitate meaningful interaction with the agencies. So when I'm doing in process testing, I have to be thinking what do I like to see during release not only in terms of critical quality attributes, but also in terms of technology. Are the technologies I'm using now for the in process testing, are they going to be used for release or not? Are to they going, are they going to be for characterization or not? Ideally, in a small biotech organization environment, I want to be able to use as much as I can from my in process characterization to release. They should be interchangeable. So for the in process it's important to have that to ensure I am actually developing and manufacturing my drug substance effectively, my drug product effectively incorrectly. The extended characterization technologies are very important to ensure if I change anything, I have good understanding of my molecule. Therefore I'll be able to check if I change a raw material, I will be able to identify that raw material has changed by doing my extended characterization. If I change vendors, I have to have an amicable extended characterization techniques to tell me I going from vendor A to vendor B, manufacturer A to manufacturer B. Therefore that comparability of Drug substance and drug product is there. Therefore we. I have to have that in place. So I'm not surprised. So I will give you a couple of examples on this one and the release panel. Normally the release testing panel is not everything in the CQA panel. It is a selected few from the CQA panel that we render to make them release assays. And that is done by vetting by also risk assessment. Some of the CQAs will remain as an in process assays. Because of the risk level, the risk may not be high. So we render them as an in process assays. Some of the critical elements of the CQA is they. They go all the way to release panel a subset of them. So these are platform release panels. So it is. It is highly strategic. Highly strategic to go with platform technology. Why is it important to go with platform technology? Number one, you save time. Instead of developing new techniques, you are using tested and tried technology. Number two, the agencies are exposed to this technology. Therefore the likelihood of the company not getting questions and information requests much lower. When we use tried and tested methods, platform techniques. And number three, the budget. You save a lot on your budget to make sure that it works for you, for the company. So these are the basic parameters and has the basic attributes. For example, for content. When it comes to gene therapy, the vector titer is very important. The infectious genome title is very important. The activities are very important. Some of this it cannot platform. Like when you for potency you have to have product specific potency. As is something. There is no platform for that. When it comes to the content. There is sort of a semi platform technology. For example, DDPCR has been used successfully for many products. Therefore it is transferable. It does require some development and optimization, but. But it can be adopted and applied freely. I would avoid new technologies when it comes to release panel as much as possible. I would rather be boring when it comes to release than to be adventurous. That's very important. Sometimes some small companies they may want to get a risk with introducing new technology. It is okay, but we need to do it in a risk assessed fashion. Identity is an important element attributes that we have to measure and the purity and impurities. All these technologies there are established basic techniques, but we also have to develop them. So we need to have a strategy on how to select platform or non platform. And of course the last part, the safety assays are pretty much most of them are compendial. We just have to use the compendium method. Unless our process does not fit these compendium techniques. And this is Basically trying to see from a vetting perspective when we have techniques is important to assess are these technologies transferable, are they complex? We need to assess their complexity. We don't have time to deal with many complex assays. We may be able to deal with one or two complex. The process is complex as it is. The number of techniques that I use for gene therapy, testing and release are double of protein biologics, triple small molecules. Therefore, we need to reduce our panel to the few meaningful assets and we need to make sure that we know what is amenable to transfer what are not. Why is that important? We don't spend too much time to transfer a complex method to a lab in a remote CMO because that may take a year and that may delay our programs unnecessarily. So we should be. We have to be cognizant what to transfer, what not to transfer. So this kind of assessment is important. It's also strategic. So this part of the selection, strategic method selection process. So we the preferred is platform and compendium is definitely as applicable. We use all the compendium available. If platform method does not exist, if we have to optimize, then we have to find a platform method and make it fit for purpose. We optimize it and develop it to make it fit for purpose. Once we do that, we assess its transferability. Because most of the time a small biotech will not be able to release in a GMP setting. May not have the GMP environment to release a drug product or a drug substance. Therefore we have to transfer it to a cmo. So we have to be cognizant about the applicability of this method to transfer. Is it transferable? Is it trainable with junior analysis in a QC setting? So that's important criteria when we develop a method and of course what's important. So lifecycle management, we have to be able to manage the method. The life cycle has to be manageable. We don't want to get in trouble. Once we have five years of this method, then we cannot get the reagent. The instrument is not supported. So we have to make sure everything we do is meaningful for a long term life cycle management of the program. So these are the criteria. As we said, we define the CQAs. Then we define the criteria for the method that we are going to use to determine the cqa. So we determine the method, the analytical criteria, analytical attribute to fit the CQAs of interest. Once we identify them, we make this method, we screen them, we screen the methods screening a method is not necessarily in the lab. It's not a word, lab screening. It could be data mining, it could be literature review, it could be assessment of what has been approved by the agency. That's what I mean by screening. But if we have the capacity and the budget, sometimes we may have to do what laboratory screening. Then of course we rank and score the technology per the need of the assessment. So this is what I just mentioned. The criteria we have, the quality attributes, we determine the analytical tools, the right attributes in the analytical tools, then we assess them and we try to define what we need, what we need and which method is more applicable for this specific scenario. Then once we choose the method, we may have to further develop it or we may be able to validate it directly. For a compendial method, we just have to do qualification for a platform method. We may have to qualify for the early phase for phase one and phase two, but then we have to validate for phase three based on the programs. So just to give a couple of example, in a form of case study, the most important, I mean some of the most critical, most important CQA's critical quality attributes are potency, identity and infectivity. So if I'm going to choose which technology is best for this, for the three CQAs, as a small company, I may want to be able to, I may want to bundle the testing, I may want to use a multi attribute technology. By that, by doing that I save money, I also save time. But the risk is that if that method does not work for one attribute, it might not work for others or there's a higher risk. So I need to assess what's out there. And for potency it's very important, that's what I'm going to deliver. And for the identity I have to make sure I'm making the right sequence, the right DNA sequence to serve the payload, the patient infectivity, that the virus will be able to deliver this payload into the right DNA machinery of the patient. This is one of the three of the most critical critical quality attributes for gene therapy. So now when I, when I select for that, I say what do I have available? I have chromatography technologies, electrophoretic mass spectroscopy, immunological assays as well as cell based assays. I have to see if I want to do, if I want to bundle them. So I need to assess each one of these, how applicable are they for each of these three attributes. It will be different for each molecule. But that assessment, that mental workout will help me navigate what is best for my molecule. So in this specific case, the chromatographic technique is good for identity, maybe activity to a certain extent, but not infectivity. The same with electrophoretic platform, the same for mass spec. But when it comes to immunological assays, I may be able to do three of them with one technology. Cell based technology may also enable me to do three of them in one. So it is a risk based assessment. And based on the assessment I choose which method I want to use in this specific case, without naming the product, the cell based assay was chosen with high complexity. In my mind, immunological might have been better. But the point is that this kind of exercise is critical for strategic analytical assessment to ensure we have the right techniques that will travel with the molecule from early to late phase. So we create method selection metrics based on what we said. For potency we need to have a criteria. Accuracy, linearity, precision, loq. For infectivity we need to look at the titer again, accuracy, linearity and precision. For identity, I need to make sure I have specific, the method has to be specific. And then how practical is this technology? If I do it in my lab, will the CMO be able to do it? That's also a very important consideration. So based on that, I create a risk matrix and also score the risk based on the risk. Choose the technology. There's a top level approach. People don't spend this, they don't spend this much effort from early programs. The idea is the earlier we do this, the more effective we are, the more success we have with the agencies down the line. This sheer exercise does help progress the programs faster. So select the analytical tools. I'll give one example of analytical tools. So another critical quality attribute, very important quality attribute is the full capsid and empty capsid. So when I have this viral capsid, it contains my DNA, my DNA is my payload. So the full capsid is what I'm looking for, full capsid is what I'm looking for. I don't want empty capsid and I do not want partially filled capsids. So this is a very important cqa and the technology to manage that, to test for this is also. We have options, but we have to be able to choose the right technology for the right setting. Because if I am not able to determine empty capsid from full capsid, if I'm not able to determine the full caps from partial field capsid, I have a risk. I can compromise my molecule, my drug substance, drug product. The empty capsids can actually increase the risk of Immune reaction immunogenicity. The partial field capsid can also if they don't have the right partial DNA in there, they could also induce immune reaction if they have fragments, if they have impure DNA in them. So having the right technique to measure the full capsids is critical. So to go through the motion. So what techniques do I have available? There are multiple techniques selected a few of them. DDPCR, Digital Droplet, PCR and UV VIS, SECMOLs IX and CDMS, CIF, AUC. These are optional technologies. All of these technologies are options that we can use for full capsid or empty capsid determination. But they have benefits and disadvantages, advantages and disadvantages. So based on this advantage and disadvantage, I will be able to select the right technique. If I'm interested only in the release panel, I may want to go with the more reliable technologies like auc. Analytical ultracentrifugation is the most widely used technique to measure full capsid empty capsid ratio. The CDM is the charge distribution Charge detection. Mass spec is also coming upcoming technique but it is not widely available for QC release use. AX is also useful, but you have to be able to develop it further. AUC does not require a lot of development. It does require tweaking and optimizing. Then you have good DDPCR analyzer, ddpc I will measure the DNA content and the ELISA will measure the protein content. And you have a ratio of these two because basically the more DNA you have to protein ratio you are on the right track. But when you use two techniques to measure to get one data, the risk of variability is high. So ideally you want to use one technique to be able to measure this important parameter. So based on what is needed, we can determine which technique is more useful. If I'm only interested for a rapid data on demand to progress a process development activity, UV VIS or dynamic light scattering may be useful. But if I'm interested to characterize I went from a manufacturer A to manufacturer B that I may want to do multiple orthogonal techniques. I may want to do CDMs, I want to do AUC and maybe DLS to make sure that I have the right full to empty ratio. So this is an example on how we can select the right technology to measure a full to empty capsid ratio. This is just basically what I said. Based on the need we could determine what kind of instrument is applicable relevant for our needs for our application. Okay, so in conclusion, so when we develop aav, having the right CQA determined early on is critical. We could call it presumptive CQAS but having that is critical because that will help us determine what kind of analytical technology are needed for the gene therapy. You are talking about 15 to 20 assay types. It's a lot of assays to manage. So the earlier the better. And part of the critical strategy is that we need to have the CKAS testing paradigm established early on. Meaning that I will need this as a release, I will keep this as a characteristic. I say when we determine that early on, it will help us minimize the burden on a QC testing down the line so we don't have to have so many assays that go all the way to manufacturing or to operation. Adopting platform technology, as I said, is important and useful. It should be determined early on. We should avoid having specified methods. Difficult, complex, difficult to transfer methods. We should avoid them unless it is really critical. Again, we should think about continuous improvement and we should think about developing the platform methods to make them far more refined for the intended use of the technology. Thank you for your attention. I have a couple of questions.

So Dr. Ness, you know, what about stability? You know, you have maybe, you know, gene therapy product or cell therapy product. How do you assess the stability of this product long term?

Very good question, Ron. So the early on, we need to make sure that by doing force deck and accelerated stability studies during early development, we established the shelf life propensity of the molecule and also determine which are the stability indicating methodologies and that we do it. The earlier we do it the better because then it will help us gain credibility on our shelf life.

When you're talking about, you know, changing some of these parameters and, or changing suppliers, you know, qualifying manufacturers, do you always, when you do that, go to potency assay to make sure the potency assay is working? Of course you have all the different CQAs, but you know, you might be standing, you know, size exclusion, chromatography might be different things. But I think the potency assay would be critical.

Yeah, you're absolutely right around. The most important parameter quality attribute is the potency. And of course in addition to that, by the risk based assessment, the full empty capsule will also be very important. We try to do, when we go from manufacturer A to B, we do a tiered approach. First we have to make sure that the release panel are intact. We also determine the new acceptance criteria, extended characterization methods. But the potency, the potency, the tighter as well as the full to empty ratio would be some of the critical elements.

Last question, Ness, in the stability studies that you conduct, are you concerned about genetic modification, where the gene might change over time or mutate or change. And how do you keep track of that? Is there an assay that you can check to make sure you have stability there?

Very good question, Rowan. Yeah, the genetic titer. So we have the right primers. When you measure for the titer, for the content, you have to amplify the right sequence of the DNA. It's a part of the stability. You measure for that, for stability, because over the years, it shouldn't change, but you have to amplify. If you don't amplify it, it will show immediately. The content will go down.

Thank you very much.

Thank you.