Fixing AAV Recovery With Siren Biotechnology’s Nathalie Clément, Ph.D.

In this episode of “Better Biopharma,” host Tyler Menichiello is joined by Nathalie Clément, Ph.D., Siren Biotechnology’s SVP of vector development. The two discuss the historical challenges of purifying AAV vectors and the developments that have allowed for greater product yields. Clément shares where she believes the greatest gains can still be made to improve vector recovery and offers advice for teams seeking to optimize downstream unit operations.

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Transcript

unknown 0:00

And and and and the film.

SPEAKER_02 0:16

Hello and welcome back to Better Biopharma, the official podcast of Bioprocess Online. I'm your host, Tyler Minichello, and on this episode, I'm joined by Dr. Natalie Clement, Senior Vice President of Vector Development at CIREN Biotechnology. You may recall from an earlier episode with CIREN's CEO and founder, Dr. Nicole Polk, that CIREN is developing a universal AAV Immunogene Therapy for Cancer, which effectively is combining gene therapy with cytokine immunotherapy. Natalie joined me a couple months ago on a bioprocess online live event with Dr. Huntrin, where we talked about downstream decisions to maximize viral effector yields and reduce cost of goods in development and manufacturing. I'm hoping today that we can continue that conversation here and go a little deeper on some of those memorable talking points. Without further ado, Natalie, thank you so much for joining me on this episode.

SPEAKER_01 1:00

Thank you, Tyler. It's a pleasure to be with you and your podcast uh team here today.

SPEAKER_02 1:06

Likewise, thank you so much for making the time. Um, and so let's just dive right into it. During that panel a couple months ago, you made the point that the industry spent a lot of years, specifically, we're talking about AAV manufacturing, because that's where you're coming from that world. Uh, during that panel, you made the point that the industry spent a lot of years optimizing upstream process development because downstream recovery was so poor historically. So I guess my question to you would be one, do you think that we're finally seeing downstream recovery improve? And moreover, why do you think that the industry is finally shifting its attention downstream?

SPEAKER_01 1:44

Yeah, so because we have a little bit more time, I'd like to maybe go back a little bit, um, a little bit further down to the past. When I said the industry has spent a lot of time optimizing upstream, it's actually pretty recently like we're talking about the past 10 years, before that, at the uh really at the birth of the AVs and the next um 10-15 years um after the AVs were becoming a really um interesting point to develop to the clinic. Um, it was upstream first, um, and downstream, kind of half and half at the beginning. Um, it's just after several years of seeing the recoveries of downstream become being so low, we're talking about a five to ten percent at the time, uh, that there was um suddenly an interest in optimizing upstream. So the the big efforts um to improve upstream is still relatively recent in considering the entire um development of AV vector manufacturing. Um and and I would say it's been the past five to maybe five to eight years that an emphasis on upstream was um really put on and developed on. Um now we've reached a point where upstream is, you know, there's always a way to optimize, but upstream is to a point where we boost uh uh AV vector yield, we boost AEV vector quality with reducing empties and residual. Um and then we've seen during that time that actually downstream yield recovery haven't really um improved much. They have improved. I know I can hear people saying, oh, we have made huge progress. We have made huge progress, but we we we are still at an overall 30% recovery at the end, at the best. So we went from a 5-10% to a 30%. I mean, it is improvement, and in terms of uh what you get at the end, uh, as we say in the field, um, every drop of AV is like gold. It's it's every drop of AV, um, uh GMP clinical material is is very precious. So we will take everything we can, and it and an increase in recovery by 10 or 20 percent is is is has a huge impact. But we can do more, right? There is still between 30 and you know the ideal theoretical achievement of 100% recovery, there's room for improvement. And to answer your question, like when um I have seen this renaissance in really trying to go back to downstream, it's it's also very recent. I I think it's really about five years.

SPEAKER_02 4:39

Okay.

SPEAKER_01 4:40

So it's kind of always been like a back and forth, yeah, until we get the balance where we we're satisfied with upstream and we think we've we we're satisfied with downstream, and then um, you know, the role of a CMC person uh at at uh or a team, right? Not just a person and at a company level is it's always when is it time to stop development? When is it time to move to manufacturing for clinical material and then and then for commercial if you if you're going through the entire uh product development um phase? Um, because you know that in six months or in a year there will be something new on the market, and there will be a better resine or there will be a better transfection reagent. So it's always uh very challenging, but also very important for a company to say, now it's the time we've invested enough in process and development, we've optimized best we could. Now it's time to stop because the patients are waiting. Um that's that's kind of uh uh uh the life of a CMC um CMC team.

SPEAKER_02 5:55

Yeah, thanks, Natalie. No, I I agree. I've had these similar conversations in the past with regarding AEV. It's like you can only chase purity so much before you're sinking a lot of time and money for little ROI. And and we'll get into that a little bit more on in terms of purity. But um, so it sounds like you're saying, is it safe to say that downstream is still currently one of the bigger bottlenecks in AEV manufacturing, if not the biggest?

SPEAKER_01 6:20

In my opinion, it is. Um, I I you know I am probably um, I would expect some people to disagree. Um, but in my opinion, it is at least I think it's where we have the most room to improvement, right? 30 to 70 percent. Um there is room for improvement, uh, which is great. Keeps people busy, keep people thinking and developing the next generation of um reagents and and material uh to implement in AV manufacturing.

SPEAKER_02 6:49

Yeah, right. I always say busy is good because busy means you're employed. So busy is never a bad thing.

SPEAKER_00 6:54

Yeah.

SPEAKER_02 6:55

Um, and I'm curious. So you said in the past decade or so, we've gone from five to ten percent recovery up to about 30% today. Is that accurate to say more or less, yes, yeah. More or less.

SPEAKER_01 7:08

I think we agree, I think we agree today, and of course, with AEV, it's kind of uh almost like a case-by-case. They are trends and they are generalities and protocols that everyone follows, but each product is different, and each company um has a slightly different protocol. So um, in general, 30% is kind of the average. Some people claim that they have more than that, and some are are a bit lower than that. So 30% is sort of agreed upon, um, expert in the field that it is where we are today.

SPEAKER_02 7:46

Yeah. And I'm curious to hear from you what technologically, what advancements kind of allowed that that percentage of youth or recovery to go up? Um, what were the biggest advancements that allowed us to go from five and ten percent recovery up to 30%? And and then further, I want to hear your kind of your spitballing on what what do you think will get us past 30%? What developments await us? Magic role. Yeah, where are we at?

SPEAKER_01 8:13

So what like they were big milestone in downstream um optimization, in my view. To explain them, I probably have to do a little bit of um again history on how new stereotype, new AAV stereotype um came came along and uh drew a lot of attention for clinical purposes. And I'm talking about the AV, you know, 8 and 9, um, very early 2000s. So before 8 and 9, the field was working with the traditional um AV2, and there were a few groups working with AV1. And I'm talking from the lens of developing clinical, clinical manufacturing, right? Not not for research. For research, there's always been a lot of uh variety in in what vector and serotype people were using, but toward developing toward the clinic, uh AEV2 and AV1 were the first one, and and they had their um their downstream processes pretty much locked down at the uh at the beginning. Uh one of the reasons was uh we knew the receptor for both AV2 and AV1, and we could develop pretty much uh immediately resins, so chromatography resin to capture this particle uh from a an upstream crude lysette. When the novel serotype 7, 8, 9 with 9 really capturing uh capturing uh the field by like excit with excitement because of tropism that had not been you know uh seen or or to the extent that had not been seen with AV2. I'm talking about muscle and and and cardio um cardiomuscular transduction. Um suddenly the field was left with there is no, we don't know the receptor of the stereotype, we don't have a resin, um a capture resin to develop and to purify this AV. So uh long story short, because they were, you know, scientists are never short of ideas of how can we purify if we don't have a good chromatography resine that is specific to the serotype, and there were a lot of um fun and powerful protocol put in place before. But the capture select, um not to say any name or or promote any vendor, but being able to develop antibody specific to the capsid of serotype 9, um, and then other serotypes, serotype 8, and then antibody that recognized um capsid from various serotypes, um that became one of the most, uh I think to me, the most impactful addition to the downstream process. This is called the capture step, which is pretty much the first real purification step that captured just the AV capsid. I mean, just uh there's always uh impurities going through, but um, these antibodies recognize form AV capsid and bound them extremely efficiently. That has in itself, this step is in itself 80 to 90 percent um efficient in in recovering the AV. So that has been a huge leap in in improving the downstream uh recovery. To that, uh more traditional but but evolved and and and optimized um onion exchange chromatography for a second step, um, more particularly for steps that um are now uh called polishing, but also enriching in full. So the capture step, which is the first step, does not differentiate whether it's a full or an empty AV capsid, just brings everything down with it. And the second step uh that is more of a which is usually an affinity chromatography, tries to separate a little bit more the full and an empty. This um a lot of um you know, better colon, better resine have been uh developed and the protocol have been tuned up to be good enough. I would say they are there's still room for improvement in there. So chromatography, big milestone, big impact. I also um is another aspect that is sometimes they are kind of the players acting in the background are the filter. Um during the downstream, there's multiple filtration steps uh to remove uh impurities like protein, um residual protein or or uh residual uh DNA and RNA. Um a lot of the new serotype we're lost, or we're binding to these filters in ways that we could not really explain. Um, and so there's been a lot of improvement on that. So altogether, I feel like we the recovery of each step individually is better, but because we still have in average four to five to six steps in downstream, the combination of the additive uh loss across all the steps still uh remain too high, in my opinion.

SPEAKER_02 13:51

I did I want to put a pin in that real quick to ask you. I'm just out of my own curiosity. I know empty versus half and partially formed capsid capture is is a huge part of recovery, and it's one of the bigger impurity aspects of of AAVs. What what step is specifically, I think you mentioned it, but I which step is specifically to sort that out to uh you know capture all the full and separate it from partially or empty capsids. And I'm just curious chemically how how does that work?

SPEAKER_00 14:24

How does that's my weak spot? Chemistry. But I can try to cut it out.

SPEAKER_02 14:30

I can try technical.

SPEAKER_00 14:32

I'm just curious.

SPEAKER_01 14:33

Yeah, I can I can I can try, uh at least I know the principle. Uh don't ask me the molecules that are involved in the interaction and and um so um I X, you know, it's it's uh it's good to spend a little bit more time on this step because it's a very complex step, and that's why even in 2026, no one really has the magic recipe on how to get from um 40% full down to 100% full. We are getting close, but it's a lot of work to get to get to that level of separation and enrichment. Um so a big bit of history also to uh sort of re-emphasize the fact that um separating and enriching, because we are calling that more enriching than separating, because we know that it's not a really kind of like empty and full. You know, the chromatogram are usually small peaks that sometimes overlap, and then they are the partial. So we are calling an enrichment step. Uh, this step is usually uh at the second chromatography step, sometimes the fourth steps after having a filtration concentration step, a capture step, which can be filtered um sometimes uh before going to the second chromatography step, which is um the enrichment in full. Um historically, that step, the separation of full and empties, was done by density gradient. Uh, you probably have heard about caesium chloride gradient, um, that they were the the first one and still are used today. They still are used today. It is still the the the golden grail of um separation of full and empty. Cesium chloride is the most power powerful iodixanol as well, um though not as often used in the clinical setting. So today the cesium chloride, iodixanol gradient are still um the gold standard in achieving a very high level of separation between full and empties, but they are so cumbersome. Um, large-scale clinical or GMP setting, um, conducting and um executing this type of gradient is is is not cumbersome and is not really prone to scalability. Um but it's still being used. So one of the big uh milestone, I believe, in downstream um efficiency and and increased recovery uh have been to implement new onion exchange protocol to separate a little bit more the empties and and the full. And you were asking me how does that work? Um it's really about playing with the charges on the AV capsid, positive, negative, and and playing with the salt and the buffer concentration to help slightly separate um the full and empty. So it's all about um charge in in the chromatography, while in the gradient it's more about size and and molecular weight. So with the empties being a little bit lighter than the full, they tend to migrate into a you know a higher um a lower density or a higher band when you're doing a gradient. So one is the size, one is charge, and it is a very simple way of explaining. Um, but that that's so to be able to separate based on charge, it's really about very tuning the buffers that you're using, the concentration in various sorts, um, and uh you know, gradient, step gradient, the magic is in there. Um, everyone does it a little bit differently, and not a lot of these protocols are published, so it's a little bit difficult to say that there is one magic rule and everyone follows it. It's not, everyone spends, I would say, years tuning up their own protocol for their own product.

SPEAKER_02 19:10

Got it. Thanks, Natalie. I appreciate you explaining that to me. There's probably some AV processors in the in the audience listening, like, we know this, Tyler, move on. But I appreciate you telling me that. Um and so back to the the downstream unit operations that you you were describing before. I want I'm curious to hear your thoughts on if improving overall recovery, is that a matter of optimizing all those steps across the board? Or do you think that there's one in particular that if if we focused our efforts here, if the industry focused on improving recovery at this specific step, it would have the biggest impact on overall recovery?

SPEAKER_01 19:54

Um in my own experience and knowing from other groups, um I think that this separation of empty full or the enrichment in full uh step, if you're doing bicromatography, of course, um, is the one where there is the biggest hit in loss. Um because as I kind of explained, there is not a really kind of clean cut between the peak of full and the peak of empties. Sometimes it overlaps, and you have to decide when do you stop getting the full until you kind of bleed into the empties. Um, so it is the biggest uh lost, in in my opinion. And so this is where you know, even improving by 30 or 40 percent the recovery at this step would have would have a big impact. Um, so that's where I would spend, and and I know you know, I'm saying this is where I would recommend people to spend time. I know this is where people spend time optimizing and tuning up. Um, so keep doing that, focus on the step first. Um spend enrichment step you're saying, right?

SPEAKER_02 21:18

The which one enrichment step, that's what we're talking about. Okay, just making sure I'm keeping up.

SPEAKER_01 21:22

And then and then a call to to reagent um developer and the next engineers is like just come up with a better resin. Or, you know, the simple, the simple solution that we've been asking for 25 years is can you just make an antibody that recognized full and only full? And then that's all capture full done. Pew sorry for this is oversimplify, it is not easy, otherwise we would have had, we would have uh had it. Um so yeah, it it is um it's it's a step that um you make your product uh purer at least certainly having less empties, but you also lose uh certain amount of your food. So it's it is where the balance between when is purity good enough versus when does it start impacting the overall um amount of material that you can generate to reach the patient?

SPEAKER_02 22:37

Yeah. Yeah, and talking about the enrichment step and separating empty versus full and this idea of pure enough is a great segue into the next part of our discussion, which I do want to talk about, you know, chasing that purity and what is pure enough. But I'm curious, just thinking out loud here, like, is there and I'm sure this has been considered before, but is there any way upstream to reduce the amount of partially formed versus empty versus like half full caps? And how much does that you know have an effect downstream?

SPEAKER_01 23:12

So it's kind of um sort of like the circle of upstream improving, then downstream bottleneck, and then upstream, uh downstream improving, upstream bottleneck. So it is really it's it's you know, upstream feeds into downstream and and upstream um respond or acts um based on what downstream um can do and the data generated from downstream. So a hundred percent upstream plays a huge role in the percentage of fool or the reduction in empties. Um, so this is also sort of back to the the first question. Why did we start uh putting a lot of effort in upstream? Was because uh we were losing so much of the good material when when we were trying to remove the empties. Um, that we're like, how about we start providing more foods? So from the beginning, so if we lose, we still end up with enough foods to go to a clinical to to enable a clinical trial. So, and that's exactly what the field did. Um, it's relatively recent. Um, this really huge focus in trying to optimize upstream, not only to increase the yield, but also increase the quality coming out of upstream. By quality, a lot is in improving the percentage of full. So, yes, um, we've made huge progress in there. Um to give you very, very average number. Uh, a traditional transfection, you know, 10-15 years ago would yield about five to ten percent full um in your uh AEV material. Today we have a combination of cell line optimization, um, suspension versus adherent, um uh new generation of transfection reagent themselves, new generation of transfection enhancers. Um, there are several uh in the market um that are boosting the production of AEV vectors and um favorizing favority, I don't know. Um, all these transfection reagents are boosting the yield up with an emphasis on increasing the percentage of full. So to share some numbers, I have seen people reporting 40 to 60 percent full from the upstream. Uh I have seen numbers higher, but I also have seen numbers that are usually more in the 2030s um percent full um in a in a in a good but standout protocol. So yeah, we've made we've made a lot of improvement. Has this helped downstream in separating full and empties? To some extent, yes. Um but it still remain you still lose um a significant amount of full when you're separating full and empties. So you just start with more, and so you end up with more, but you're still losing. So there is still room for improvement.

SPEAKER_02 26:38

Yeah, sounds like there's room for improvement on both sides, but particularly, yeah.

SPEAKER_01 26:42

Absolutely.

unknown 26:43

Got it.

SPEAKER_02 26:44

And you know, what we talk about, uh purity was a huge part of the our live event panel some time ago, and so during that event, we we asked the question, what is pure enough? And I want to ask you, do you think the industry has become too focused on maximizing purity as opposed to recovery, or is there no such thing? Because I know obviously purity is tantamount to safety, which is the ultimate goal when you're going into patience, but at what point is is pursuing higher and higher purity not worth the squeeze, as they say.

SPEAKER_01 27:17

Yeah, this is the one million dollar question. Uh, and and I cannot answer um, this is enough pure, let's move on, or we need to keep working on on purity. I think the best way I can answer that is pure enough when it's safe enough. Um, and it could be first of all, there is no such a number of like pure, right? So in the attribute or the certificate of analysis of a clinical AV material, there is one test called purity, and you're getting a percentage, right? Has to be more than 90% pure, but that's just one test based mostly on um protein purity. And then you have all the other tests that are measuring the residual DNA, residual benzonase, residual everything, which are component of a purity assessment. Uh, and we have yet, as a field, to combine all this measurement into one number that would tell us okay, our PrEP is 99.99% pure, considering all these uh impurities. I think probably we should start thinking about that. Um this put aside, um it's a case by case, it's uh it's really about what is your CMC scale look like, what is your uh commercial manufacturing plan look like, and it's looks like, and it's very different if you're going for uh an ocular disease or a systemic disease like Duchenne uh Duchenne muscular dystrophy, right? In one side, you are adding like 10 to the 11, 10 to the 12 um vector genome, and on the other side, four log higher, 10 to the 15, 10 to the 16, and closing to closer to the 10 to the 17. It's it's a huge difference. Um so if you're going with a large-scale bioreactor, but your end product is going to be filled at a very low concentration for a very small volume to be administered in your patient. I feel like you have more room to add steps to if if you if you want to reach a higher purity. Um, and if you feel like higher purity is uh necessary to make your product safer, uh when you hit a very large-scale manufacturing, every drop of AV is critical, but the safety as well, right? Because you are starting to administer very large amount of your uh drug material drug product, and and and purity comes um also as something extremely important. So looks like I'm saying, yeah, whatever, I'm not answering really the question, it's really a case by case, and purity is assessed during your toxicology um studies, um, which should tell you if your product is safe enough to be an um to start a clinical trial, and then your human uh uh data are you know helping kind of understanding and assessing your your your product safety, and then adjustments are made um based on that. So yeah, this is where I'm gonna leave it. Yes, if uh purity very important, but maybe let's not, you know, we should not chase the holy grail, especially when we develop a product for first time in human, um, until it's actually proven to be beneficial into a uh into a patient. Um I feel like sometimes people try to make it too good very early on, and and that delays uh the product to reach patient uh in a timely manner.

SPEAKER_02 31:38

Yeah, that's great. I was gonna ask you how you think indication and dosing requirements affect a product's target purity. And I mean, it sounds like you kind of captured that and saying this case by case, unless you can't.

SPEAKER_01 31:50

But I want I want to make clear that every single product, whether it's for an ocular disease or or uh a systemic disease, uh metabolic disease, they are still screened and scrutinized and assessed the same way, right? There is not a preference for one versus the other. I hope that didn't come this way.

SPEAKER_00 32:08

Of course.

SPEAKER_01 32:09

They are still scrutinized by the same quality attributes, um, and they are still the general rule of what uh residual level should be or not, and what purity should be. Um, I was just trying to say that it's it's it's a bit more challenging at large scale to implement additional steps that could add a little bit uh more purity without um impacting the overall recovery.

SPEAKER_02 32:37

Of course. Yeah, I think you I think you perfectly captured it when you said unless you could prove that there's a verifiable benefit to patients in in achieving higher purity, then you know, just meeting those requirements is whatever gets you to the next developmental phase, right? Whatever gets the product to move forward and ultimately helping patients. So that was a great answer. I think you were.

SPEAKER_00 32:58

You got it, Tyler. Great.

SPEAKER_02 33:01

Thanks, Natalie. Thank you. Um, I did want to talk about kind of downstream, looking at downstream design as a whole. And so again, I keep referring back to the live event. If you haven't watched it, it's available by a process online, fully recorded. Uh shameless plug right there. But during that during that event, we talked about kind of combining unit operations to to to simplify downstream and kind of achieve more with less, right? And so uh pulling on the thread a little bit, I want to ask you a hypothetical here if you inherited a process that had super poor recovery, just pitiful, where would you first look to improve it? And I guess we'll keep it downstream. Downstream, where would you first look to improve recovery?

SPEAKER_01 33:47

Um, so first I would want to understand why is there a poor recovery by asking, you know, basic question. Is it a new stereotype? Is it something that no one else has ever produced before? Do we have all the reagent and all the tools uh commercially available to be successful? And so assuming it's a stereotype other people have done, they are the reagent and tools in the market to play with. Um then I would of course look at each unit operation individually, look at the one that there is a larger loss. Um, if you have a loss during filtration or TFF, there's something that you're doing that's not correct. Um, and it's usually easy to fix. So I would look at that. So the TFF are like sort of like filtration and concentration at the same time. Um, it's usually done either once or twice during your downstream process. Usually, if you start with a very um you know large volume of your upstream, we're talking 500, 600 liters, 1000 liters, right? You want to kind of concentrate this uh to make it a little bit more um easy to to handle on the downstream. Um and then I would look at your capture step. Um again, if they are uh if there is um a resin in in in commercially available for a zero time that is not out of the you know ordinary, um, you should also get at least 80% recovery. So if you're not there, start looking at it. And it's usually about the buffers and the steps and and and the um the washes and and and all of that. It's really the buffer composition that make make a big difference. Um, and I bet you that it's gonna be into your enrichment step. So closing the loop on that, um, probably start focusing on that and explore alternatives to what you are doing or or improve the steps that you are currently doing.

SPEAKER_02 36:01

That's great. Thank you. Thank you for answering that hypothetical. And again, like I said, during that event we talked about combining unit operations. Yeah, I'm curious what do you think, which unit operations do you think can be most effectively combined or eliminated altogether? And I'll make it a two-part question. Which unit operations deserve the most attention in the name of uh optimizing recovery downstream for AV?

SPEAKER_01 36:25

And I think I don't remember exactly how I said it during the the live event, but combining unit operation is sort of like my uh my motto. It's like I want to combine unit operation, I want to do two in one, I want to, you know, reduce the I won't, I won't for the field, of course. Um you know, reducing the number of steps so that you know, removing some of the small losses that are all around this small step and and combining into one, it's easier, it's faster, it's more efficient. Um, so I would I would think it's something that I probably said. Um again, I would say the one or the two unit operation that would love to combine it's the capture where you are capturing uh most of the AVs and the separation full and empty. So right now it's two different uh uh it's an affinity chromatography for the capture step based on antibody, and then it's an onion exchange chromatography for the separation uh step. Um combine them, uh, capture and try to capture mostly the fool. Again, it sounds easy, but it's and I'm sure people have worked on that and and are still working night and days on that. Um we're not there yet. I bet you that we will find that one day. I don't know when, but yes, so that that would be the two unit operations that I would combine. Uh the other unit operation to sometimes reconsider whether they are really necessary, um, are the the filtration steps and the filtration and concentration step, we which are TFF. Um although usually the recovery is pretty good, there's always loss associated with with a filtration by just even just you know the volume that's um that that's lost or retained within the filters. Um it may not be all the time necessary to have filtration in between each steps. Um, so I would I would challenge teams that have um filtration steps at each step or major filtration step to kind of really look at what are you gaining um beside you know beside sterility and making sure we are you know not carrying on um advantageous uh agent and and and bacteria, of course, this is important, but besides sterility, um I should not stay sterility, people would say sterility is at the end. So during the process is more like bio-burden, like making sure there are no uh nefarious agent being going through the the system. Does it add something into your purity? Do you remove residual when you're filtering? If so, how much how much protein, how much DNA could you skip a few of these steps without impacting your chromatography stuff? Um, yeah.

SPEAKER_02 39:55

Great, thank you. That was a great answer. I appreciate that. Um, okay, and now moving on to the the last part of our conversation, just closing questions here. Um, curious to hear from you what are you excited by right now in the in the field of AAV or uh downstream innovation? Just whether there's new technologies on the horizon. You described a couple ways you hope to see um downstream improve as far as recovery, but is there anything actively happening right now that you are excited by, Natalie?

SPEAKER_01 40:24

Um, yes, of course, I'm always excited to see any kind of progress. Um I'm excited now, although it's it started about you know a few years ago, but I was so happy, so relieved and excited when I started to see people really trying to understand the AV biology during production. Understanding and and that was so I'm bringing you back to upstream. Um this has been a big um relief when I uh when I when so money, resources, and and expertise being uh utilized to optimize upstream to understand that you can, you know, and and again, my second motto, if there's two motto for me, um people who work with me kind of have heard them like so often that they are like ah is look at the virus, look at the AAV wild type virus. How does it get to produce so much uh particles? How does it interact with the cellular machinery? How does it interact with the helper virus, which we do not have in a vector um manufacturing platform, but the white-type virus needs an adeno-associated or an hairpiece um uh virus to replicate. Um, and so look at that, understand that better, understand the kinetic of rep and cap, uh, the the two proteins that are required to replicate the AV vector genome, and then to package this genome into a a capsid, look at the kinetic, the level of expression, and try to fine-tune your process to really look more like a wild type virus. And and people have done that, and and it has made significant improvement in the upstream. Uh, and I'm excited to see um you know, vendor now offering, you know, there's different names like enhancer, um, all all of these new molecules that are commercially available really are impacting the kinetics um of the production of the AV vector in the cell by impacting the cell cellular machinery, the the replication, and so forth. So there's a lot of science behind every new thing, and and and there's more to come. So that I'm very excited, and that should continue on the downstream. Um, excited about the progress that we've made in separating full and empty by chromatography, because I, you know, as much as the cesium chloride gradient are efficient um at separating full and empty, um no one really wants to bring a caesium chloride gradient to a commercial level uh manufacturing process or to a thousand or two thousand-liter um scale manufacturing process. Although it's done, no one really wants it because it's really cumbersome and risky. If you run fans, you you kind of lose a lot of material. So excited about the focus on the second step that is enriching fools by onion exchange chromatography. But I Have not seen recently, haven't seen this this year or last year anything really, really new in in how we approach downstream. I'm waiting. So I'm waiting to be blown away. I'm waiting to be excited again by by really kind of inventing something different for downstream. Um that could that could that could benefit everyone. So I'll let you know.

SPEAKER_02 44:33

Yeah, please do. We'll have to update. We'll have to run it back. We'll we'll have an emergency podcast taping if something huge happens. That's great, Natalie. Thank you. Um, and this last question here, it's a question I ask every guest on the show, and that is big or small, whether it's related to what we're talking about or something else entirely. Natalie, I'm curious to hear your thoughts on how you think we can better biopharma.

SPEAKER_01 44:59

The only request I have is to make it more affordable. So why I'm saying that is you said biopharma, I'm thinking, you know, and the end goal is patient being treated with this amazing uh biological product that we are developing and spending years developing and spending years, you know, fine-tuning, manufacturing, making them safe, making them powerful and efficient. So the end goal is to get into the patient. We all know that even the the few marketed products that reached patients are still um are very expensive. Like I think there is no other word. They are like the one two million dollar uh treatment. Um it's it's even in the you know mainstream media, everyone knows about it. Uh and I think lowering the cost um starts with lowering the cost of manufacturing. We all know that CMC uh manufacturing, testing, which we haven't discussed today, could be a whole other podcast on that. Uh it's extremely, extremely expensive and time consuming, extremely expensive. Um, and it's a big portion of the final price tag. So better biopharma means making things in a way that it reaches the patient and and it is affordable for everyone, just not you know few ones that the families have to do uh fundraising and and and going everywhere to ask for money to be able to save save their children. Uh it's beautiful, but we should not have to do that. So this is where I think biopharma um should focus on with the patient and affordability in mind.

SPEAKER_02 47:13

I completely agree. I couldn't agree more, Natalie. Thank you. That's a great point and a great note to end on. Thank you so much for joining me on this episode of Better Biopharma, the official podcast of Bioprocess Online. If you want to hear more, head on over to Bioprocess Online for the full recorded live event with Natalie and Hung, where we discuss downstream decisions to maximize viral vector recovery and reduce cost of goods. As always, I'm your host, Tyler Menichello, and I'll see you next time.