Case Study: Sustainable Plasmid DNA Strategies

Elisa Manzotti (00:06):
Well, hello and a very warm welcome to today's Cell and Gene Therapy Insights webinar on sustainable plasma DNA strategies, achieving streamlined, secured supply to clinic and commercialization. I'm Elisa Manzotti, founder of BioInsights and joining me today are Dr. Andrew Frazer, who will explore some of the common challenges encountered when sourcing plasma DNA and provide valuable recommendations. And Amanda Weiss who will present a case study and highlight key lessons learned to help developers set themselves up for success when working with CDMOs. So after our presentations today, we'll have a Q and A session and do feel free to pose your questions to our presenters using the ask a question box at the bottom of your screen and we'll try to get to it during the session. And if your question is for a specific presenter, please just try to include their name in the message.

(00:53):
And so now I'd like to introduce our presenters, excuse me. Dr. Andrew Frazer is the Associate Director of Scientific Solutions at Charles River Laboratories. Since earning his PhD in biochemistry from Queens University Belfast, Andrew has attained over 10 years of experience in the design, implementation and tech transfer of processes for the manufacturer of biocatalysts, small molecule APIs and biologics. In his current commercial role, Andrew focuses on the Charles River, excuse me, plasma DNA manufacturing platform eXpDNA, and Plasmid CDMO service offerings. And joining him today is Amanda Weiss, the VP of CMC, Purespring Therapeutics, a kidney gene therapy company. She has over 27 years of industrial experience in the development manufacturer and analysis of biological products and has held posts at both product development companies and CDMOs. She trained as a biochemical engineer at the University of Birmingham. Well thank you both very much indeed for joining me today. Without further ado, I'll hand over to Andrew to start today's presentation.

Dr. Andrew Frazer (02:18):
Okay, so hello everyone and thanks for joining. So I'm struggling with a little bit of a cold today, so please bear with me and excuse the sniffles, but it does give me great pleasure today to have the opportunity to speak to you in this joint webinar between Charles River and Purespring Therapeutics where I'll begin with a brief introduction to Charles River and a little bit about plasmid DNA and its importance in advanced therapy applications. And then I'll pass over to Amanda who'll discuss Purespring and some of the amazing advancements that they're making in the field of kidney disease. I'll then pick up and close out with an overview of Charles Rivers recently launched eXpDNA plasmid platform and how we've progressed this service offering to support our client needs.

(03:20):
So it was a very brief introduction. I expect many of you listening and involved in the development and delivery of medicines will probably at some point have had some interaction with Charles River. The company has a very extensive history and also worldwide presence with over 110 facilities supporting all stages of drug development. And this goes all the way from research, discovery and safety assessment and right through to manufacture and testing. We're very proud to have been directly involved with the development of 86% of novel FDA approved drugs in 2021. And this actually includes around 1300 INDs or IMPDs with about a third of these within cell and gene therapy.

(04:14):
While Charles River is well known for its history and also the capability and testing, in recent years through a number of strategic acquisitions, the company has established a global CDMO network and this spans each of the major CDMO platforms. It includes cell therapy and viral vector at our Baltimore, Memphis and Rockville sites in the US and plasmid DNA production at our Keele and Alderley park sites in the UK. So when we combine CRL's extensive capability and capacity and testing with the experience and expertise from our new CDMO sites, it has allowed us to put together a strong foundation to enable an end-to-end portfolio of services for the advanced therapies.

(05:04):
So now a quick overview on plasmid DNA, plasmid now plays a very important role within the modern healthcare sector. The market is significant with estimates in the range of 4 to $600 million. An estimated compound annual growth rates at around 20% continuing from 2022 and through to '23, reflecting growth in the wider advanced therapy space. Plasmid DNA is already in high demand and it's clear that the growing adoption and acceptance of advanced therapies utilizing plasmid DNA, both as a direct therapeutic product or as a critical starting material for onward manufacture is driving increased demand. And this in turn is creating challenges and also opportunities for suppliers and consumers.

(06:04):
This slide shows the major uses of plasmid DNA for advanced therapies, on how plasmid can be used on its own as a direct therapeutic product in the case of naked DNA therapies or plasmid vaccines. But importantly, it also occupies the role of a critical starting material for onward processing for many different advanced therapy applications. It's important to note that in the development and delivery of a medicinal product, any of the building blocks that are shown here on this slide rely heavily on the one proceeding it. So it is critical to have a very safe, reliable and also cost effective supply and reliable supply to deliver effective treatments to patients.

(06:51):
So hopefully that short intro gives you a feel for Charles River as a company and also the important role of plasmid DNA for advanced therapies. I'll now hand over to Amanda to talk about Purespring therapeutics and the work that they've been doing on kidney disease supported by plasmid supply from Charles River.

Amanda Weiss (07:22):
Thank you Andrew. I'm just about to share my screen with you all.

(07:33):
Okay, so thank you for that overview, Andrew, of plasmids. I would like to introduce Purespring to you. So I'll spend the next few slides telling you who we are and where we're focused. So we are leading a revolution in kidney disease, we're the first gene therapy company that has a platform and we're aiming to deliver locally, by local administration. Our target is the podocyte cell in the kidney, and this is founded on the science from Professor Moin Saleem from the University of Bristol. We have a number of programs underway, we have three programs at the moment and we have quite a powerful state-of-the-art search engine called FunSel, but I won't focus on that today.

(08:30):
Purespring was set up about two years ago with funding from Syncona just shy of $60 million and we've got a great quality team with experience of taking products all the way through to the market.

(08:44):
So kidney disease, this is a huge unmet medical need. Roughly one in eight people around the world suffer from chronic kidney disease and this has an impact on the cost of treating and looking after these patients. And it's predicted that the number of deaths from kidney disease by 2040 could well be an excess of 3 million people a year. So it's really a huge area that needs more attention.

(09:15):
So why do we focus on the podocytes? Well, the podocyte is a terminally differentiated cell and so it's a great target for gene therapy. It's also implicated in about 60% of renal diseases. So there's many manifestations that can go wrong in the podocyte leading to different diseases. It's part of the glomerulus and it has a number of functions within the kidney including regulation of filtration, structure of the kidney and things like regulation of complement modulation.

(09:55):
Because we are targeting this single cell and because it has such a wide reaching implication in a number of diseases, we can build a platform around this. So this enables us to use the same capsid, a Rep cap and promoter and as I mentioned earlier, have a targeted route of administration. And that results in a lower risk for us, reduced cost of goods and will help us accelerate our timelines because we can leverage the platform for different indications. In addition, this platform also applies to the CMC because we can use common process to make the vectors and this offers us cost benefits, but also means that we get experience with a baseline process, so that allows us to have reliability and reproducibility in the process. And it also means that we can expose a process to the regulators that after our first indication they will be familiar with having seen it.

(11:04):
So I talked about local delivery. This could be a real big advantage for us because we're targeting the kidney directly, it would allow for optimized efficiency, potentially a lower dose would be required and that would impact cost of goods and also limiting systemic exposure because rather going intravenously and systemically around the body, we're just targeting that organ.

(11:40):
So a little bit more about our process. I don't think there'll be too many surprises here. So we use a triple Transient Transfection into Serum Free Suspension Cell Culture and these plasmids we get from Charles River. The quality of these plasmids is really important to us we believe, because by having a consistent high quality feedstock, it allows us to build a process as I mentioned, that we can get robustness from and reliability. So I'll talk a little bit more about plasmid supply and working with Charles River in a minute. And then as I said don't think there's too many surprises here. Our process follows on with primary recovery for clarification, a capture step, [inaudible 00:12:27] enrichment, trying to predict the regulatory field in introducing adventitious virus removal and then UFDF followed by formulates and fill.

(12:43):
So at Purespring, there's a number of us here that have been in CMC for a long time and through that history we've learned good things to do and not so good things to do. So it's from this that we've been able to build a number of principles that will help us de-risk our delivery. So these principles include solid foundation, thinking about the regulatory and quality environment, so the quality of the raw materials that we're looking to supply, how we can de-risk delivery. So working in partnership with people such as Charles River and having flexibility, working with those, and leveraging technology platforms that we can adapt and innovate and between us learn from shared experience.

(13:37):
So working with Charles River, they help underpin a number of these principles for us, helping us future-proof our investments, offering full GMP, having a great partnership with them, them being flexible and as I said, sharing that experience and learning from that experience together.

(14:00):
As a small business we have quite aggressive clinical development plans and so our starting materials are absolutely critical, it's building those foundations to our success. And as I mentioned, we are adopting our CMC principles here to safeguard our future development and reduce our delivery risk. So we do outsource our plasmid supply, we start off from the very beginning, manufacturing GMP cell banks. We are trying to anticipate the regulatory environments by doing this because we believe that by having the same source of our plasmids for both R&D plasmid and for our first in human plasmid, we will have less potential changes in the future.

(14:55):
As I'm sure many of you're aware, there are many plasmid vendors out there and we did our due diligence on a number of those vendors and we selected Charles River and the main criteria that we selected them on was because they do offer full GMP, they have a very comprehensive quality system and are able to release plasmid to us directly. Had good capacity and speed at which they could address our needs. Working with the team, they're very supportive and very flexible. We talk about if slots need to change or be put back or accelerated, they do their best to try and accommodate our requests. We have a great partnership with them, it's a very open relationship, interactive and indeed they've demonstrated, previously Cobra, they have a great track record and they're very experienced in plasmid DNA manufacture. So Charles River do help us adhere to our principles and I've just again highlighted those things that where we work with them and they help support de-risking our delivery.

(16:16):
So just to summarize really when we're setting up and thinking about critical starting materials and who we're going to use for those starting materials, think ahead of how you want to... Try and forward think how you're going to be successful. And that's about building a solid foundation, thinking about quality and regulatory very early on, working in partnership so that they can help de-risk your delivery and looking at technology platforms out there and how you can adapt to them. And as I said, identifying those partners that can deliver all of those attributes that help build those foundations and CMC principles for us to be successful. Okay, thank you. Back to you Andrew.

Dr. Andrew Frazer (17:12):
Thanks. So hopefully everybody can see my slides again. But yeah, thank you very much Amanda, it's always fantastic for us as a manufacturer of plasmid, particularly when plasmids are often supplied to our customers as a starting material for lots of onward manufacturing steps and often plasmid can be quite far removed from the final product. So it's great to see how our products are used when they go out there to our customers and hopefully that provides some valuable insight to everybody listening.

(17:56):
The next part of I guess my section of the presentation is to follow on and talk a little bit about Charles River's recently launched eXpDNA plasmid platform and this has been set up directly to address some of the most common plasmid supply challenges that we've encountered in recent years with a focus on delivering both exceptionally good timelines but also with a very strong focus on process and product quality attributes.

(18:27):
So the eXpDNA platform is, as Amanda mentioned, it is a development of our longstanding plasmid service offering, and that was previously Cobra biologics. And it has been developed based on three core capabilities. So the first is a plug and play toolbox approach where we can adopt various levels of screening, quality control checks, excuse me, to mitigate the manufacturing risks that are associated with often complex and challenging plasmid types that are commonly used for advanced therapy applications. The second is a standardized platform manufacturing process, which allows a streamlined supply chain and documentation management. And last but very certainly not least, we now being part of Charles River and the wider Charles River network, we have the ability to implement 100% in-house analytics for both cell banking and also final product release testing. So by integrating these three capabilities, we can deliver industry leading batch turnaround timelines to our clients while maintaining a solid focus on quality.

(19:54):
At the center of the eXpDNA platform, our plasmid facilities. So [inaudible 00:20:00] Charles River established our dedicated Plasmid DNA Center of Excellence in the UK. So our Keele site, which was previously Cobra, has been producing both GMP and non GMP plasmid products for over 20 years and is now a fully dedicated GMP plasmid manufacturing site and it's currently undergoing significant capacity expansion. Alderley Park is Charles River's newest CDMO manufacturing site and it opened in October last year. The site has three fully segregated and independent processing streams that can all operate in parallel to deliver HQ plasmid. And it utilizes process facility and product quality specifications that are aligned to principles of GMP for high quality plasmid DNA manufacture.

(20:53):
I guess we'll finish just by saying that development of these sites represents a significant expansion to Charles River's CDMO manufacturing capability for plasmid and it is a very important step as the company continues to respond to industry feedback and deliver an integrated global network to support the delivery of advanced therapy products.

(21:18):
So as mentioned, our plug and play development toolbox plays an important role within the platform and as Amanda highlighted in her presentation, the cell bank is of critical importance to lay a solid foundation for any plasmid or equally any onward viral vector manufacturing program.

(21:37):
Our cell banking process itself and the testing is very well established and it has been in operation for a long time delivering cell banks for plasma products right through R&D to commercial supply. And in doing this, we have developed an effective approach to efficiently and robustly screen different plasmid types that are commonly used in advanced therapy applications. And this allows us to navigate many of the common issues that are associated with the generation of reliable plasmid containing cell banks.

(22:13):
So following an initial technical assessment of the plasmid sequence, our team will identify the type of construct and any key features that are critical to its function. This will then I guess inform our approach for the subsequent cell banking steps that we need to include and dictate which additional screening or quality control measures that need to be included to ensure a very smooth transition or progression through manufacturing testing and release. So there are many plasmids that can progress directly through manufacturer with minimal checks or screening. However, some of the more challenging types like ITR containing gene of interest plasmids for AAV or messenger RNA template plasmids containing long poly-A sequences, they typically involve some extra steps to fully manage and mitigate their unstable nature or I guess propensity for recombination during cell banking and manufacture.

(23:22):
So the Ambr 250 system that we have is an effective tool for early stage screening and pre-production evaluation and it's used routinely to provide an early indication on process performance, also productivity assessment and yield prediction. And this type of assessment can be implemented on its own but also in parallel with pre banking and clone selection steps to confirm plasmid integrity using methods like NGS sequencing or genetic stability testing. And this system has a very strong track record in managing these challenging plasma types. And with the rapid in-house testing that we have in place, we can implement this approach for our customers with relatively little impact on overall project cost and timelines.

(24:23):
Our plasmid production process also follows the same principles as our cell banking approach with a focus on rapid turnaround and also retention of end product quality to deliver a right first time manufacture of plasma products. The process is not plasmid specific, but it has been designed to accommodate a wide range of different plasmid types without the requirement to perform extensive and time consuming optimization work. Both our main production processes for our high quality and GMP plasmid service offerings now operate with a fully single use process stream and this includes dedicated single use pre-packed chromatography columns and materials and reagents that are free from animal origin.

(25:15):
And through the delivery of many different plasmid types over the past 20 years, we can be confident of predictable scale up and importantly reliable product specifications and retention of stable plasmids through the full manufacturing process.

(25:32):
I'll finish just again with a little bit more on single use processing. So it does carry a number of benefits. When operating particularly multi-product facilities where we're often progressing parallel manufacture of a range of different plasma products for different customers. One of the main advantages for us as a manufacturer is that we avoid costly and time consuming equipment cleaning and residuals testing between batches, but also the fully single use process stream effectively rules out the possibility of product cross-contamination. So our customers can have full confidence that the risk of cross-contamination has been fully mitigated.

(26:23):
While there are clearly advantages to running fully single use processes, those involved in the bioprocessing industry will I'm sure have experienced challenges particularly since COVID around supply of single use components [inaudible 00:26:42] with lead times for things like bioreactor bags and tubing manifolds often on the critical path for manufacturing project timelines. And while suppliers of single use products are making their own improvements and this lead times are improving, it is something that Charles River recognized. And as a response within the eXpDNA platform, we have put significant effort into standardizing our process and materials and also led leveraging partnerships with our global suppliers.

(27:18):
It's a very important development in our platform and it has allowed us to simplify our supply chain and hold larger stocks of materials, which effectively allows immediate initiation of manufacturing projects and avoids issues related to long lead time consumables. This slide summarizes our core plasmid DNA service offerings with again, a focus on industry leading timelines and product quality. So our high quality or HQ grade plasmid represents a more cost-effective step up in quality from R&D grade plasmid rather than going directly to full GMP. While it's sometimes used at a very early stage, it's generally adopted in some applications for toxicology studies, also as a critical starting material for GMP vector production in phase one or two clinic and also as a template for GMP messenger, RNA production. It incorporates a range of GMP principles and it uses fully traceable materials, a comprehensive documentation package and it's produced in dedicated and segregated HQ production suites with delivery of custom plasma products in as little as five weeks.

(28:45):
GMP products are manufacturers to the highest possible standards in our licensed GMP production facility at Keele. We have fully comprehensive documentation, testing and quality assurance oversight. And as Amanda mentioned, we also have onsite QP release. Our GMP plasmid is the gold standard and it can be used at all stages of clinical supply and commercial viral vector manufacturing and also DNA vaccines. And due to the advancements within the new eXpDNA platform, GMP plasmids can be delivered in a 10 week turnaround.

(29:26):
One final point to add and one that we're very excited about at Charles River is the launch of our off the shelf packaging and helper plasmids for AAV and lengthy viral vector applications. And these will allow effectively immediate supply for both HQ and GMP grades and offer significant time and cost benefits for wider cell and gene therapy programs.

(29:56):
So I've talked quite a bit about our platform and standardization, some of the benefits that it can generate with regards to speed of delivery, but I thought following Amanda's presentation and some of the points you touched on, it would be appropriate to look at some of the different options that developers have with regards to plasmid quality grades and importantly some considerations for cell banking. So if we look at the three common plasmid quality grids that are available, R&D, HQ and GMP, they all require a plasmid containing microbial cell bank to be produced before production and purification of the final plasma product. So in the case of R&D plasmid, we start with a research cell bank followed by research grade plasmid manufacturer. And because of this, these products are relatively low cost and they can be made quickly and with streamlined quality standards and documentation and they're typically used for R&D or preclinical applications.

(31:07):
HQ can be produced using production cell banks with HQ grade manufacturing to deliver material for tox in some cases and critical starting material for viral vector manufacturing. In this case there is a significant uplift in quality and this comes obviously with some additional cost and time to manufacture. Finally, when we look at GMP, this utilizes full GMP master cell banks. All GMP manufacturing processes are performed inside licensed and qualified clean rooms. And this generates plasma that can be used for clinical and commercial applications. And this is obviously the very highest level of quality but comes at the highest cost with the longest lead times. And within the eXpDNA platform, our customers have the option to implement any of our screening tools within any of these approaches to ensure reliable production. However, if we are going to go down that route and like Purespring have done invest upfront and work to future proof their plasmid products and supply, it is worth looking in a bit more detail around some of the options and considerations that you have for cell banking.

(32:30):
So as we mentioned, the quickest and cheapest option is a research cell bank followed by research grade plasmid. However, it is important to recognize that it's not possible to utilize research cell banks for HQ or GMP grade plasmid manufacture. And the transition later on to increased quality grades can be very challenging with regulators. The additional manufacturing costs and also the time to establish new cell banks later on to deliver higher quality plasmid, those need to be factored in and considered for longer term supply requirements. And developers also need to be fully aware of requirements for comparability studies.

(33:17):
Adoption of HQ grade production cell banks provides a little bit of a middle ground and developers can realize some cost and time savings and I guess flexibility around r&D supply if they wish to use these cell banks to generate R&D material in addition to any HQ grade manufacture. But production cell banks cannot be used directly to produce GMP grade material in the future. So it's important to note while I'm saying this, that for some applications where the plasmid is for example, a critical starting material and it's separated from the patient by subsequent GMP manufacturing steps, for example in cell therapy, there are scenarios where the use of production cell banks and HQ grade plasmid can satisfy supply requirements right through to commercial. However, there is always the risk present that regulators will tighten up on requirements and developers will then need to go back and establish a GMP supply.

(34:30):
So what we're seeing more and more now is that many of our customers, including Purespring, are investing in GMP grade master cell banks at an early stage in order to provide high levels of flexibility and that they can transition easily over to HQ and R&D plasmid supply if needed. It gives a strong foundation of quality at a very early stage and it's a way to future-proof your supply and mitigate the risk of any regulatory changes or having to incur comparability studies that could have very major impacts on the time and cost of advanced therapy programs.

(35:17):
So to close out, firstly wanted to extend a big thank you to Amanda and her colleagues at Purespring. Purespring is a fantastic company to work with and I expect to see them have every success as they progress the amazing work that they're doing and make a real difference in the treatment of kidney disease. I also wanted to thank the cell and gene therapy insight team for putting this joint webinar together. I hope everybody listening found it useful and that we've helped to highlight some of the strategies that can be incorporated to support plasmid supply for advanced therapy programs and aid the delivery of these life-changing medicines. Thank you.

Elisa Manzotti (36:06):
Fantastic presentations there. Thank you both very much indeed. And so we have time now for our Q&A session, so we've had some questions coming in already from the audience, so I'll get cracking with those. So our first question is, I know that speed is very important for startups as you've mentioned here, but how does choosing an MCB over PCB impact timelines or time to clinic? Andrew, I was going to come to you here first.

Dr. Andrew Frazer (36:32):
Sure, so yeah, it's a good question. I think we touched on it in the presentation. I think first it's important to recognize or appreciate there're loads of contributing factors that influence plasmid supply options for different companies depending on what stage they're at, what funding's available. Time and cost are very major ones, particularly at an early stage with an unproven product or technology. And it's very important to try and understand I guess for those who are making those decisions, what options are on offer and plan ahead and hopefully webinars like this help with that. I think what I would say is that if a customer knows early on that they will at some point definitely need to make the transition from HQ to GMP plasmid, we'd always recommend going for an MCB as early as possible as we can use that to produce HQ grade material or even RD grade material very easily versus a PCB.

(37:40):
In addition, I would say avoiding comparability studies and also the additional manufacturer of an MCB at a later date is very, very significant with regards to long-term projects, time and cost. So that would be my main, points to make around the choice of cell bank, MCB or PCB.

Amanda Weiss (38:05):
Yeah, I think I can add a little bit to that. So I think one of the biggest hurdles with any contractor is signing your MSA and quality agreements, which can take some time. With respect to speed, okay, the GMP cell bank does take a little bit longer to manufacture, but as Andrew's quite nicely made the point a number of times, you've got a consistent starting material that can feed the production of your R&D grade plasmid if that's what you want to use and that's exactly what we do and we feed that into our process developments and then we can use that same cell bank to make GMP plasmid for our tox and first in human studies. And the reason we do that is because we know that we have got the same source material.

(38:55):
If we were to make an R&D cell bank, particularly with the GOI, we're dealing with quite repetitive sequences in the ITRs. So if we were to do a new transformation in GMP, we could see a deletion or a mutation as part of that transformation. So then the material you generate from that could be different from the material that you generate from your R&D material. So by making your master cell bank early and using that as the source of all your starting material, you know you've got... And you can also put it on stability as well early and start generating stability data on that. It just helps future-proof your future process and the plasmid that you are using for your development and clinical studies, and that's exactly why we do that.

Elisa Manzotti (39:45):
Fantastic answers, thank you very much indeed. And I suppose that sort of ties in with additional questions you received regarding how you mentioned you chose GMP plasmas for your first in human studies. What other sort of additional factors were feeding in there to why you chose the GMP grade over high qualities?

Amanda Weiss (40:02):
Again, we go for GMP grade, because we use that in our tox and our first in human trials. The regulators don't necessarily stipulate that, but again, because we want our tox readouts to be indicative of what the human studies may or may not look like and look at safety and so on, again, that's another driver, it's about being consistent, having those same starting materials. And as Andrew's mentioned many times, and I've seen this, if something goes wrong or changes, then you want your starting material to be a factor that you can rule out that's not implicated in a change or an adverse event.

Elisa Manzotti (40:48):
Yeah, that makes a lot of sense. Andrew, do you have any points to add there as well?

Dr. Andrew Frazer (40:53):
No, I think Amanda has more than covered that.

Elisa Manzotti (40:59):
Fantastic, thank you. So we have a question that's coming from Steve. Do you foresee a continued demand for principles of GMP plasmid or do you see an increase in cost pressures on full GMP grade? Andrew, I'll come to you back.

Dr. Andrew Frazer (41:18):
Yeah, so I think it's an interesting one, so there does seem to be an increase in the level of testing and quality control standards around HQ grade plasmids. And I think the useful thing at the moment is that... Because the regulatory advice around this, so obviously with DMA delivering articles around guidance, on the principles of GMP, it does help manufacturers frame that better and it gives us a much better, basis to plan and deliver on our manufacturing activities with regards to application, I think if we do see this continual tightening of regulations around testing and manufacturing, it might be easy to say that eventually it'll transition totally to GMP and intermediate grades like HQ will be phased out. That being said, GMP plasmid is very expensive and one of the challenges with advanced therapies is their costs.

(42:39):
So I'm sure everybody is aware of some of the costs associated with licensed advanced therapy products and having very complex manufacturing approaches and materials involved in the delivery of these therapies does contribute heavily to those costs. And I think manufacturers are working on that. We're doing lots of work at Charles River on reducing cost of goods, but equally having the ability to utilize a phase appropriate supply like HQ grade and using principles of GMP to risk assess the potential impact to patients and adopt those intermediate grades and that phase appropriate approach also helps to drive down those costs and timelines as well. So from that point of view, I think if we do see wider adoption of... An acceptance by regulators with some of these products where we are seeing HQ grade go right through to late stage clinic and even into commercial supply, we could see it continue for a long time.

Elisa Manzotti (43:56):
Yeah, absolutely. Amanda, did you have much sort of engagement on the regulatory side of things? I was wondering if you had a perspective on whether you feel there's a movement towards the requirement of GMP rather than high quality?

Amanda Weiss (44:05):
Yeah, it's a good question. I mean, if more and more people do use GMP grade as critical starting materials and the regulators see that, then I think they sort of adopt those practices. So it's always the highest standard that they like to see. Again, the reason we do it is to future-proof our process and where we're going. And although yes, it's expensive, but again, if we end up having to go into a comparability exercise and plasmids is one of those things that we have to investigate the time and cost of doing that could... particularly if you are going for a rare disease indication and you don't get exclusivity to the market because you're delayed, it could cost you your products. So it's a good point with high grade, high quality DNA being used. But again, as I say, we are aiming to future-proof our future program.

Elisa Manzotti (45:08):
Makes a lot of sense. Thank you.

Dr. Andrew Frazer (45:11):
I think there is a lot of regulatory... It's a bit of a regulatory gray area around some of these intermediate products and like I say, the additional guidance is helping, but it is very, very application dependent. For AAV, absolutely, the vast majority of our customers are moving towards GMP when they get into clinic. I think it's some of these cell therapy applications where the plasmid is used as a critical starting material, you've got multiple kind of GMP manufacturing steps and product release in between and it's very far removed from patient and that's where we see the longer term application of HQ grade DNA.

Elisa Manzotti (46:00):
Great answers. Thank you both very much indeed. Moving on to our next question. So realistically, what are the real world advantages of being a single source provider, Andrew?

Dr. Andrew Frazer (46:11):
Okay, so yeah, I think we did touch on it in the presentation. It is something that is within Charles River's long-term plan and we are moving very quickly towards that goal with bringing on these new CDMO manufacturing sites, that's added a big piece. So I think with regards to real world advantages, it's very hard to give a firm answer. As I've said, every project, every product is different, but particularly for advanced therapies, there is published data to show that there's an increase of issues and complications with CMC when... And a much higher disruption for regulatory approval versus more traditional products like [inaudible 00:47:04].

(47:04):
And it's something where we feel that an integrated project approach from a single source provider can help to more rapid delivery by increasing some of the efficiencies that we have throughout the development process. So things like the quick turnaround times that we talked about with the eXpDNA platform allow effective and timely decision-making. Utilizing a single experienced and multidisciplinary team supports, I guess more effective use of data for project progression, also helps with problem solving. And one of the most important things that we can do, for example, having your plasmid and your vector production under one roof is to align your manufacturing timelines and testing with study activities to optimize scheduling. So like I say, it is difficult to give an answer. I think there's huge opportunity and scope to improve in this regard, but certainly the advantages are clear and they're there to be had.

Amanda Weiss (48:26):
Yeah, I think also it helps the client out potentially because if you have your AAV manufacturer looking after your plasmid supply, they need to ensure that they've got that plasmid readily and available to start the AAV manufacture. So it does help with logistics and timing and release of those critical starting materials into your vector supply. So as a small business for us who outsource a lot of activities, it's not so much less to manage, but it's putting it together, which does help with managing and logistics of plasmid supply.

Elisa Manzotti (49:08):
Thank you. Okay. And then looking at our next question, so besides lead time, what are some of the other advantages of off the shelf plasmids? Amanda, I was going to come to you first though. Or Andrew, do you want to go first?

Amanda Weiss (49:24):
Well, maybe Andrew can...

Dr. Andrew Frazer (49:26):
[inaudible 00:49:29]

Amanda Weiss (49:29):
Andrew's start and I can chip in.

Dr. Andrew Frazer (49:31):
Sure. I mean, lead time is obviously a key benefit, but I guess from a manufacturing point of view, having some of these commonly used plasmid types available, it helps reduce pressure on our manufacturing and testing capacity. I think also the ability to be able to generate these plasmids in larger bulk quantities, you kind of incur all the advantages of economy of scale. So it allows prices to be reduced and also customers then have the opportunity to plan ahead and place orders for their programs on a kind of a gram basis versus on a batch basis. And I think anybody who's got experience in sourcing plasmid knows that the different plasmid types, you do get variability around batch to batch yields, it's difficult to predict sometimes. And certainly being able to order quickly on that kind of gram basis would help with avoiding issues with under, over ordering and wastage.

(50:46):
I guess also when you think about this further, there's probably lots of secondary benefits around reduction in complexity of supply chain. So something we talked about a little bit about your vector supplier source in plasmids, the ability to reorder at short notice. But on top of that, I think if we could standardize the use of some of these common packaging and helper plasmids, then it does have the potential to ease regulatory approval and also the introduction of follow on pipeline products for developers in the longer term.

Amanda Weiss (51:28):
I think I would add to that as well. So as with any product, whether it be starting material API, you have to put it on stability. So our bespoke plasmids, we have them all on stability, which means that you have to dip into your batch and sacrifice an element of your batch to put that on stability. And it may be that you have two or three batches on stability. So for me, being able to buy it off the shelf, particularly with things like helper, then I would refer to CRLs DMF as part of our regulatory filing with the manufacturer of that, so they would support the stability and the storage conditions of those plasmids, which actually saves me a bit of time and money.

Elisa Manzotti (52:14):
Very important consideration indeed. Great. Okay, I think we have time for one last question that's coming from Thomas. How are you defining the specific differences between the RCB and PCB? Is that testing or documentation? Andrew?

Dr. Andrew Frazer (52:28):
So RCB to PCB, yeah, there are some key differences. So yes, documentation and testing are two. So documentation for RCBs, generally we're working from a manufacturing protocol. It wouldn't be what you would consider to be a batch manufacturing record, like what you'd see for an HQ process or a GMP process. Also, the level of quality oversight is very much reduced compared to high quality or obviously GMP. Testing panels are typically heavily reduced. Customers can request to have any of our GMP tests included for a research cell bank, for example, but typically they're not interested because the material's not high value, the level of application, they don't need that much coverage. So yeah, the testing is significantly reduced. Although at Charles River we do use exactly the same assays, so our customers do get that continuity, if they want to transition up in the quality grades, the testing should still hold and it's easy to do those comparability studies if they're needed.

(53:46):
On top of that, the manufacturing environment is probably the biggest difference. So for research grade, effectively it's done in a research laboratory, so minimal control of environment, we would use traceability on materials, we record things like [inaudible 00:54:08] numbers, but you wouldn't have the same processes and procedures that we would have for HQ. And then obviously GMP taking that a number of steps further. So those would be the main differences between RCB and then PCB, which kind of falls in the middle between a GMP master Cell Bank.

Elisa Manzotti (54:32):
Thank you very much indeed. So that just brings us to the end of our Q&A session. Thank you so much, Andrew and Amanda for great presentations today, and I also thoroughly enjoyed our discussion. And thank you to our audience, as always, for your questions, there are a few that we couldn't answer during the live session, but please rest assured that the Charles River team will reach out to you directly after the webinar to respond to those queries. And just as a reminder, the [inaudible 00:54:55] webinar is available tomorrow, so look out for an email from me and do feel free to distribute it and share it with your colleagues.

(55:03):
Well, thank you again, Andrew and Amanda, it's been a pleasure speaking with you today and to our audience, I hope to see you again next time. Thank you very much indeed.

Amanda Weiss (55:08):
Thank you everybody. Bye-Bye.

Elisa Manzotti (55:11):
Bye.

Dr. Andrew Frazer (55:11):
Thank you. Bye.

Amanda Weiss (55:11):
Bye.