How to Future Proof Your Plasmid Supply and Avoid Common Pitfalls

Sarah Hiddleston (00:00:02):
Hello and welcome to this Nature Research Custom Media webcast titled, How to Future-Proof Your Plasmid DNA Supply: Avoiding Common Pitfalls for Clinical Trials and Beyond. My name is Sarah Hiddleston and I will be your moderator. Today's webcast is sponsored by Charles River.

(00:00:24):
Improving levels of success and a large number of advanced therapy products at late phase clinical studies mean there is an ever-increasing demand for plasmid DNA supply. In addition, evolving and sometimes undefined regulatory requirements and quality standards present a range of opportunities and challenges for developers. In this webcast, we'll hear from Dr. Andrew Frazer of Charles River Laboratories. He'll explore some of the common challenges encountered when sourcing plasmid DNA, and provide valuable recommendations to help navigate common pitfalls and future-proof your plasmid supply to support clinical programs.

(00:01:07):
We'll then move on to a question and answer session with you, the audience. You can ask a question at any point you wish throughout the webcast. To do so, please type your question in where it says, "Type your questions here" and then press submit and we will answer them at the end of the session today.

(00:01:27):
And now, over to Andrew.

Andrew Frazer (00:01:34):
Hi everyone, and firstly thank you for joining today. I'm going to talk about supply of plasmid DNA and hopefully provide some useful guidance and also considerations for drug developers, particularly at an early stage within the product lifecycle. We'll look at some of the things that we can do, both as suppliers and also consumers of plasmid DNA to help future-proof the plasmid supply and avoid common pitfalls, and ultimately fast track the delivery of new advanced therapies to patients.

(00:02:12):
So first of all, a very brief introduction to Charles River. I'm sure many of you will have interacted with the company at some point as it has a very long history, and really a worldwide presence with over 110 facilities supporting all stages of drug development. In fact, Charles River was directly involved with the development of 86% of novel therapeutics approved by the FDA in 2021, and this included around 1300 INDs and IMPDs with around 30% of those being within the cell and gene therapy field.

(00:02:53):
While Charles River is very well known for its history and capability in testing, we've also now established a global network of facilities spanning all of the major CDMO platforms. And this includes cell therapy and viral vector at our Baltimore, Memphis and Rockville sites in the US, and also plasmid DNA production at our Keele and Alderley Park sites in the UK. So when we combine this manufacturing capability with the extensive capability and capacity afforded by our testing sites, we can see a true end-to-end portfolio of services for the delivery of advanced therapy products. And, this goes all the way from discovery and nonclinical development right through to GMP manufacturing.

(00:03:48):
So hopefully that very brief introduction helps highlight the current position of Charles River within the wider advanced therapy space. But for the remainder of the presentation today, I'll be focusing on plasmid DNA, and in particular the key role that it plays in supporting the development and delivery of advanced therapies.

(00:04:10):
I'll start with some backgrounds on sourcing and supply of plasmid DNA, mainly to highlight some of the challenges that we face both as suppliers but also as consumers. And after that, I've selected two focus areas that I feel are important to plan properly and get right. And again, the focus is on an early stage within the product lifecycle.

(00:04:34):
The first is the establishment of plasmid containing cell banks, and the second is considerations for the selection of different plasmid quality grades. Both of these are common areas where we can implement relatively simple strategies and risk mitigation measures at a very early stage to help future-proof our plasmid supply. And this will help support right from the early stage through even to commercial product manufacturing.

(00:05:07):
So this first section will look in general at plasmid DNA sourcing and supply, and as I mentioned, highlight some common trends and complexities. I think it's fair to say that plasmid DNA is firmly established and it plays a major role within the modern healthcare sector. The market for plasmid DNA is significant and estimates in the range of $400-$600 million, and then that was from 2021. And the projected growth of that market reflects the growth in the wider advanced therapy space with compound annual growth rates around 20%. 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 manufacturer of viral vectors or messenger RNA, or even transient proteins is certainly driving up increased demand. And in turn, this creates challenges as manufacturers try to keep up with supply requirements.

(00:06:25):
This slide shows this in a more visual manner where we can see how plasmid DNA itself can be the direct therapeutic product, and this would be in the case of naked DNA therapies and vaccines, but importantly, it also shows how plasmid products occupy the key role of a critical starting material. And this spans many advanced therapy modalities. It's important to recognize that each of the building blocks shown here relies on the one proceeding it, and ultimately everything in the chain has to work well in order to deliver safe and effective treatments to patients. If we consider the common role of plasmid DNA, it highlights the importance of getting it right from the start with your plasmid supply and the importance of laying a solid foundation for the development and delivery of advanced therapies.

(00:07:23):
This slide shows some data taken from Nature Reviews and Drug Discovery. In this case, they were looking into disruptions in the development of wider cell and gene therapies. We can see in this example that unlike classical antibodies, lots of advanced therapies are having issues and complications that lead to clinical hold and delay, also discontinued regulatory submission and approval. And when we compare this directly with mAbs in this example, we can see that cell and gene therapy products experience a much higher rate of disruption. And this ultimately drives up cost and time on development programs en route to market. If we look at the root cause analysis within this report, we can see that many of the issues stem from manufacturing and comparability between clinical right through to commercial supply. So, it does highlight a need to address CMC issues and the requirement for stronger validation and continuity when we progress from development through to clinical trials.

(00:08:40):
If we keep this in mind, when we look at common sourcing and supply strategies for plasmid DNA, it's clear that there are many areas where we can encounter these types of issues. So if we broadly group developers or consumers of plasmid DNA into three main types, we have academia, who are typically involved with product discovery and development, often producing their plasmids through their own research groups or using specialized academic centers. And, these same academic centers often also supply emerging and sometimes well-established biotech companies with early stage supply plasmids through development and preclinical. And it's really at this point when we start to have the first point to crossover with different types of contract manufacturing organizations, or CMOs, supplying everyone really, so academia, biotech, and right through to large pharma. And it's generally when large pharma move towards market supply for certain types of licensed products that plasmid DNA manufacturing goes in-house to larger purpose-built facilities.

(00:09:56):
So, if we look at how this potentially plays out for an example product moving through discovery clinical trials and then to market, we can see that there are many points of transfer. And this would include information, data, even physical material exchange between multiple entities. And this includes academic groups, multiple CMOs, and potentially then having to tech transfer or perform parallel development of commercial manufacturing processes in-house under a regulated environment for a very well-defined and established product towards the end of the lifecycle.

(00:10:41):
So changes are generally very, very difficult to make. And with this in mind, it's therefore very important that we do everything that we possibly can at an early stage to try and avoid the types of disruptions that we previously discussed, so issues that are common when transferring supply from one place to another. And also think ahead as far as possible to make the transition from development to preclinical into clinical trials, and then finally, market supply as easily as possible.

(00:11:17):
So one of the key requirements for plasmid manufacturing, which can have a very positive influence if it's done well, or equally it can create significant impacts to time and cost if it's overlooked, is the generation of cell banks that are used for the production of plasmid DNA. When we look at plasmid DNA production approaches, there is of course variation with different manufacturers, and this depends on available equipment, different production scales, utilization of specialist technology, but they do all follow a general framework. And in contract manufacturing, manufacturers need to accommodate multiple plasmid products and often multiple host cell lines for production, and the processes are often set up in such a way that they easily deliver productivity within acceptable ranges for lots of different products while still maintaining commercial requirements and delivering effective cost of goods for consumers.

(00:12:24):
In addition, CMOs want to fast track development studies and optimization work. And they do this because they want to deliver quick manufacturing turnaround times and also contain project costs. But before the actual plasmid manufacturing can take place, a suitable cell bank needs to be manufactured, and this is done under control conditions with full characterization and testing.

(00:12:52):
And to briefly run through, a single vial of this cell bank has then progressed to manufacture where it's expanded in a bioreactor. It would be induced to produce high plasmid copy numbers per cell and then those cells are harvested. The harvested cells would be lysed open to release the plasmid product and then progress through a number of different purification steps. And this could include multiple chromatography steps, different filtration steps basically to remove any host cell debris and other contaminants that we wouldn't want in our final purified plasmid products.

(00:13:32):
So looking a bit closer at cell banking, cell banking follows three main steps. So firstly, a small company of plasmid is transformed into a characterized host cell line, successful transformant, so then selected by antibiotic resistance. And finally, acceptable clones are expanded before the addition of a cryopreservant and then banked for long-term storage to support onward manufacturing needs.

(00:14:04):
Where we can run into issues, particularly within early stage development is on the host cell line selection. So for example, cloning strains that are used for the development of plasmid constructs in research and development may not be suitable for GMP manufacturing purposes in the future. Or equally if laboratory strains are used, they may not carry sufficient information on their origin, evidence of controlled handling or quality control information to enable direct transfer to a manufacturer.

(00:14:42):
One of the main aims with any cell banking process is to generate a homogenous population of cells containing the correct products, the methodologies that are used and also the level of QC checks that are implemented at the clone selection stage. And also the cell banking stage itself can have a significant impact on the quality and also the reliability of the banks that are intended to be used for manufacturing. And in the case of some common challenging plasmid types that we'll go on to look at in a bit more detail, critical issues can go undetected for a long time if they're not properly managed.

(00:15:32):
Finally, the quality of information captured on storage of plasmid cell banks, the testing methodologies that are used to characterize them, and then the certification that is provided can also heavily influence the options that early stage developers have when they want to transition through the various stages of preclinical and clinical plasmid supply.

(00:15:59):
So where these issues become particularly apparent is when we start to consider challenging plasmid types that are commonly used for advanced therapy applications. So a really good example of this are ITR containing plasmids that are used for a adeno-associated viral vector or AAV manufacturer. AAV production requires three plasmids, so we have a packaging plasmid, a helper plasmid, and then a transfer plasmid. And it's the transfer plasmid that contains the therapeutic gene of interest. The gene of interest plasmid has a well-established configuration and amongst other things that are critical to its function. The gene of interest is inserted between two inverted terminal repeats, or ITRs. And these ITR regions are critical for replication and packaging of the recombinant genome during vector production. And also the function of the ITR regions is critically important if we are to achieve consistent purity and potency of the final AV product.

(00:17:10):
Unfortunately, when we propagate these plasmids during cell banking and production, the ITR sequences are inherently prone to deletion due to the second restructure and also high GC content. To make things even more challenging, ITR deletions are not reliably detected by simple analytical methods like restriction digest. So it is possible to progress through the clone selection stage during cell banking without picking up these types of deletions unless further measures are taken.

(00:17:51):
Additionally, standard Sanger sequencing methods that are typically used to assess plasmid identity and integrity following completion of cell banking typically struggle to sequence through ITR regions, and we can easily find ourselves in the scenario where we have a transfer plasmid cell bank containing a completely dysfunctional transfer plasmid. Or, what's even worse is where we've generated a mixed population of functional and non-functional plasmids that can be very, very hard to detect and it can lead to per and also inconsistent AAV production.

(00:18:32):
What makes this even more challenging is that if we consider that the development of these plasmid types, and certainly the manufacturer and supply of this type of plasmid, is usually running in parallel to the development of an AAV production process and the associated analytical testing, an undetected issue like this, it can take months or even years of onward development work to troubleshoot and resolve.

(00:19:02):
So I've tried to provide a little bit of insight into some common challenges and also that clear example with the ITR plasmids. And, these are challenges that we encounter directly as a manufacturer of plasmid DNA and also through feedback and experience with working with a wide range of drug developers.

(00:19:23):
So what I'd like to do is just talk a little bit about Charles River's recently launched eXpDNA plasmid platform. And this has been set up firstly to deliver exceptionally good timelines, but also with a strong focus on process and products quality attributes. And this, again, is in a direct response to help address some of these common challenges that we see.

(00:19:51):
Following the previous section, I hope there is an appreciation for the importance of a high quality cell bank, and within our plasmid platform at Charles River, we do view the cell bank as the foundation of any plasmid manufacturing program. While our cell bank process is very well established, it has been an operation for many years, we've also developed an effective and robust set of tools that allow us to efficiently screen a range of plasmid types that are commonly used for advanced therapy applications. And this allows us to navigate many of these common issues associated with the generation of reliable plasmid containing cell banks.

(00:20:38):
The first step in initiating any plasmid manufacturing project always involves a technical assessment of the plasmid sequence. The outputs are the identification of the type of construct and how we handle it based on previous experience as well as any key features that are critical to its function within its intended application.

(00:21:03):
It's probably worth saying before we move on that there are lots of plasmid types that would not require any of the additional screening or quality control measures that are shown in this slide. However, some of our more challenging plasmid types, like the ITR containing plasmids that we looked at earlier, or for example messenger, messenger RNA template plasmids containing long poly(A) sequences, they typically involve some extra steps in order to manage and mitigate the risk of failure when we reach final product testing and release.

(00:21:38):
So the Ambr250 system that you can see on this slide has been in operation at our Keele site for a number of years, and it's proven to be an effective tool for early stage screening and also pre-production evaluation. We can perform up to eight fermentation runs in parallel with this with a very rapid turnaround. And, this gives us an early indication on how plasmids will perform within our manufacturing process.

(00:22:04):
It gives us a productivity assessment and also a yield prediction so that we can plan ahead with our plasmid supply. This type of assessment is often performed in parallel with pre-ranking and also screening steps that involve the selection and actual banking of a number of clones. And this is followed by checks with various analytical methods. And our go-to really for these challenging plasmid types is NGS sequencing. So when these are implemented, it does provide full confidence that the individual clone that we select as part of the screening process within cell banking is correct and contains a stable plasmid product, and allows us to generate reliable cell banks for production.

(00:23:00):
It's a system that we have had in place for a number of years and with the in-house and rapid turnaround testing that we have from the Charles River network, we are in a position where we can recommend and implement this approach for our customers with relatively little impact on overall project cost and timelines. So for some of our challenging plasmid types, it is a very worthwhile investment at early stage.

(00:23:28):
In addition to this cell banking approach and the robustness that it brings with selection of reliable cell banks, our plasmid production process also follows the same principles. And it again has a focus on rapid turnaround and retention of end product quality to deliver right the first time manufacture. Our production process is not plasmid-specific. In fact, it is quite the opposite and it's been designed to accommodate a range of plasmid types without having to take on costly and time-consuming optimization work. Through the delivery of many different plasmid types over the past 20 or so years, we can be confident of predictable scale up and most importantly reliable end products, specifications and retention of plasmid integrity through the full manufacturing process.

(00:24:27):
Both of our main production processes for high quality and GMP plasmid now operate with fully single-use process streams, and this includes dedicated single use pre-packed chromatography columns. To compliment this, we've also put significant effort into standardizing our process and materials, and this has allowed us to simplify our supply chain and we can now hold larger stocks and material, which avoids issues related to long lead time consumables that are really a common challenge across the whole bioprocessing industry.

(00:25:09):
Importantly, and one point that it is important to discuss is that utilizing a fully single-use process stream does carry a number of benefits particularly as a manufacturer, when we're operating as a multi-product facility manufacturing a range of different plasmid products for many different customers. So one of the main ones for us as a manufacturer is that we avoid costly and time-consuming equipment cleaning and residuals testing between batches. And this allows us to quickly turn around production suites and use them more efficiently.

(00:25:43):
But also and very importantly for our customers is that a fully single-use process stream effectively rules out the possibility of product cross-contamination. And when we combine this with our cleaning and segregation procedures, we can have, again, full confidence that the risk of cross-contamination and the catastrophic impact it can have to wider programs has been fully mitigated.

(00:26:14):
So now I'd like to present a couple of real life case studies to highlight some of the strategies that we can implement to manage common issues and the value of taking a quality focused approach on cell banking.

(00:26:30):
So case study one is from a very experienced developer within gene therapy, and after having success with the previous product, they now wanted to fast track manufacturing of plasmids to support their AAV pipeline products. So they had a range of ITR containing gene of interest plasmid candidates that they wanted to progress in parallel, and they were well aware of potential ITR stability issues. So through development of the plasmid constructs themselves, they had incorporated a commonly used restriction digest check for ITR integrity, and they wanted to invest upfront in full GMP grade master cell banks. And, they required purified plasmid to support AAV manufacturing for both toxicology and early phase clinic.

(00:27:27):
As I mentioned previously, they had very aggressive timelines. There was a requirement to parallel track activities but also take all possible measures to avoid any manufacturing issues that could lead to repeats or introduce significant delays on their wider program.

(00:27:44):
So in this case, the customer did have a well established relationship with another third-party supplier to provide starting plasmid that they could send to us to enable GMP cell banking and onward plasmid manufacture. And I think it's an important point to note that this starting plasmid did come with testing certification to show ITR integrity by both restriction digest and sequencing. And again, based on having done this lots of times, in order to fully mitigate the risk of carrying forward an undetected ITR deletion through to the MCB, we included pre-banking of 12 clones as part of the master cell bank production. And interestingly, even though the starting plasmid was confirmed to have intact ITRs, we did pick up deletions in 60% of the clones that were tested by next-gen sequencing. And this, again, highlights the importance of using high quality analytical checks at multiple points through the process of establishing your cell bank.

(00:28:57):
With these checks done and with a successfully transformed clone with an intact construct, we're able to progress through to final MCB manufacture, and to support the timelines that this customer had, multiple vials were allocated for non-GMP use and the manufacture of high quality grade plasmid for toxicology. And, high quality grade is something we'll talk about a little bit in the next section.

(00:29:27):
But following the full MCB release, we were then also able to go on and provide GMP grade plasmid for clinical use.

(00:29:39):
The next case study is ... it's quite a different situation, but as you'll see, it does provide some useful insight into again, common cell banking issues and also the importance of proper management. In this case, we again had a well established company and they were in early stage development with a number of candidate plasmids that were going to be used as template for linearization and then onward, messenger RNA manufacturer. They had gone through multiple years struggling with a polyA chain deletion, which is common to this sort of plasmid, and they couldn't get to the point of generating acceptable cell banks when working with other CDMOs.

(00:30:26):
So at this point they were exceptionally risk averse and wanted to take every measure possible really to deliver a reliable GMP cell bank that they could then use for their development work, but also manufacturing of template plasmid right up to even commercial production skills.

(00:30:50):
So in this case, we implemented a staged approach with this time extended clone screening and analysis. So stage one included selection of 30 clones followed by restriction digest and sequencing. And out of the 30 initial clones that were selected, we observed about a 90% success following restriction digest, and this was simply to check for a known recurring deletion outside the polyA tail. We then selected 10 of these successful clones for sequencing to assess the polyA tail retention, and about 50% of these returned intact sequences.

(00:31:39):
Stage two involved a genetic stability screen by serial subculture, and this also included a temperature shift that was representative of the plasmid production process. So in this case, out of the five clones that we progressed from stage one following the screening by restriction digest and then sequencing, we actually achieved 100% stability of the transformed plasmid to up to or above 60 cell generations in this. When you get to that level of cell generation, it's probably representative of manufacturing up to multiple thousands of liters, so really suitable for any scale of production that would've been required for this sort of product.

(00:32:22):
Stage three, we moved on to the Ambr250 system that we discussed earlier, and we used this to assess product quality through a representative small scale manufacturing process. And this provided an opportunity to perform some high level process optimization, but it also generated a batch yield prediction. And at this point we also included end of production cell bank testing with further sequencing to confirm the polyA retention.

(00:32:52):
Finally at this point, we were able to progress to MCB manufacture and deliver GMP grade cell banks that went on then to support future manufacturing requirements for this customer.

(00:33:09):
So to summarize on plasmid cell banking, it does represent a critical step in the manufacture of plasmid DNA, and because plasmid DNA is so important for lots of different advanced therapy products, it's an important one to get right. And it is clear that some of these challenging plasmid types that are used commonly for advanced therapy applications, they can't be overlooked. And in order to future-proof your supply, additional quality control checks are often required. Restriction digest is definitely a useful and commonly used tool for screening. It's also used as a release testing assay for identification purposes. But for some plasmid types, if it is used to check some of these challenging regions, it does need to be backed up by other methods like next-generation sequencing.

(00:34:12):
Across the board really, we are seeing a trend, particularly with more experienced developers where they're investing in GMP grade master cell banks at an early stage to support all of their future manufacturing activities in this. It doesn't necessarily mean that they commit to GMP grade plasmid at early stage, as even for large companies that would be cost prohibitive, but it does mean that they've made a relatively small investment upfront and it provides then the option to easily swap over to GMP plasmid in the future if the need arises.

(00:34:53):
Finally, there are very few programs that don't require transfer of materials, or at the very least information between manufacturing sites as products progress through clinic towards commercial. And these transfers can be challenging depending on different procedures and safety requirements at different sites. So planning ahead as far as possible is important, and I think it's important to ask yourself, "Am I getting the correct information and data that I need from my current supplier to allow me to have that flexibility in the future?"

(00:35:36):
So the second topic that I'd like to discuss is different plasmid quality grades and when to use them. There are a number of different quality grades of plasmid DNA that are commonly used to support advanced therapy applications, and these are sourced by developers based on the phase and the production scale, which is related to the amounts required for a particular application and also the compliance or the level of quality.

(00:36:04):
So on one end, we have research grade plasmid, which is mainly used for R&D or preclinical. It's produced in an R&D laboratory and it comes with very simplified documentation and a reduced panel of analytics. And this is mainly to reduce time and cost.

(00:36:19):
The next step-up is high quality or HQ grade plasmid. This does represent quite a big step-up in quality from R&D, and while it's sometimes used at a very early stage, it's generally adopted for toxicology studies or as a critical starting material for GMP vector production in phase one or two clinic. It can also be used as template for GMP messenger RNA production.

(00:36:46):
In this case, we're into fully traceable materials, much more comprehensive documentation packages, and it's also produced in dedicated and segregated production suites utilizing GMP principles. And this is something that I'll talk about in detail in the next few slides.

(00:37:05):
The last and the highest quality grade and also the most costly is GMP plasmid. This can be used basically for anything. So clinical supply, commercial vector manufacturing, and also DNA vaccines. GMP products are really manufactured to the highest possible standards. This is performed in licensed GMP production facilities, fully comprehensive documentation testing, quality assurance oversight, and also QP release.

(00:37:40):
So as an advanced therapy developer, how do you decide which grade of plasmid to use and when? To help answer this question, I think it's worth looking back quickly at the history of plasmid DNA regulatory guidance for advanced therapies.

(00:37:59):
It's fair to say that historically it has been challenging to clearly identify exact specifications and quality attributes, and this has made it difficult for both manufacturers of plasmid and also end users to make clear decisions on the development of drug products. So in 2005, the EMA issued guidelines for the production of lentiviral vectors, and this included a reference to plasmid being high quality. Unfortunately, the definition of high quality was not so clear and manufacturers largely determined specific details themselves.

(00:38:35):
And as a result of that, we saw the launch of different HQ plasmid service offerings or equivalent service offerings from different manufacturers, and developers had to basically provide justification for its use with regulators on a case by case basis. The 80% covalently closed circular specification provided by the FDA in 2007 for plasmids that will be used for direct human use, for example, the DNA vaccines has been retained as I guess the minimum industry standard for all plasmid DNA products, but with many manufacturers also producing supercoil plasmid now with much higher specifications.

(00:39:20):
In 2018, the EMA guideline defined plasmid DNA as a starting material rather than a raw material and added further guidance around ID testing. A particular note would be the requirement to fully sequence the therapeutic gene of interest. And additional guidance was then also provided by the FDA in 2020 for manufacture and testing of plasmids for gene therapy.

(00:39:48):
So at this point, I think it's still fair to say that high quality grade plasmids fell within a regulatory gray area, and with a gradually evolving set of guidelines, it was quite difficult for manufacturers and also end users to clearly nail down their quality requirements depending on their application.

(00:40:08):
And on top of this, there was always the risk that any further regulatory updates or changes in approach regarding the use of intermediate grade plasmids like HQ could result in an impact to either ongoing or future development of the drug products that were reliant on plasmid production.

(00:40:30):
So to build upon this, I want to highlight two points of reference that became available in late 2020 and early 2021. So, the first is this BioPhorum article, and it provides a review and discussion summary on plasmid release specifications for both E. coli cell banks but also purified plasmid products. And, the aim of this article was really to set out and work towards a consensus platform approach for plasmid products by essentially consolidating feedback from lots of the major players in the plasmid manufacturing world. It is an interesting article. It does provide some good insight into common trends and practices within the industry and also how things are evolving from a regulatory standpoint.

(00:41:28):
I've included some points in this slide that highlight some of the, I guess, the inconsistencies that are present on plasmid release specifications and methodologies. And, these include identification tests like restriction digest and sequencing, which we've already talked about today. We've already talked about some of the limitations with traditional Sanger sequencing methods and also the emergence of next-generation sequencing as the go-to for challenging plasmid regions. But certainly in the future, we need to consider if the method, the coverage, and even the quality of sequencing reads needs to be the same for all plasmid types or even specific regions within plasmids.

(00:42:13):
Again, methodology is evolving for DNA homogeneity or percent that's supercoiled, but certainly the level of accuracy and the inclusion of other analytical outputs. For example, different plasmid DNA isoforms may be dependent upon final application. Endotoxin is an interesting one as there is significant variation on target specs that are offered by different plasmid suppliers. So I think as a consumer or as a drug developer, it's very important that you ask yourself when selecting a plasmid supplier, or even a grade of plasmid from a supplier, to consider the potential impact of higher endotoxin levels, both for onward processing, but in some cases patient safety.

(00:43:09):
Sterility and bioburden. Testing are established as I guess common prerequisites for onward viral vector manufacturing. And this also includes things like mycoplasma testing, and particularly if we have to transfer the plasmid to a separate manufacturing site. But in the future, we will see plasmid DNA starting materials moving more towards rapid methods like BACT for sterility or PCR based testing under a risk-based approach, particularly when the plasmid product is far removed from the patient.

(00:43:57):
The second reference, which has a significant influence over the future of intermediate grade plasmids is the 2021 EMA Guidance Document reviewing principles of GMP and where these apply in the manufacturing of advanced therapies. So this document provides some clear regulatory guidance on quality standards for plasmid used for all major advanced therapies. And the approach that was provided is not that surprising, and it's one that many manufacturers were already adopting.

(00:44:28):
Essentially the end product, whether that's a naked plasmid, a viral vector, or modified T-cells, the closer it is to the patient, the greater the level of quality oversight that's required. And the main takeaway is that GMP grade plasmid is not required when used as a starting material for advanced therapy products, but for certain biological materials, it is mandatory that we apply and adopt GMP principles. And the term GMP principles simply means that manufacturers of advanced products need to ensure that appropriate GMP requirements are implemented for manufacturing and testing of starting materials. And, we do this using a risk-based approach.

(00:45:17):
So as a manufacturer, we now had a much more clear guidance and opportunity to align our high quality plasmid service offering while at the same time also progressing fit-for-purpose solutions to some of the new customer regulatory requirements. So we developed a HQ service offering that adopted principles of GMP. We introduced full flexibility around the use of our cell banks, also incorporating with this a fully animal free production process, which is now aligned also with our GMP production process.

(00:46:03):
As we talked about a few minutes ago, the manufacturing process itself is fully single-use, which effectively manages cross-contamination. And our HQ and GMP plasmids now also come with the option of fully aligned testing and also addition of optional sterility and mycoplasma testing.

(00:46:25):
The last piece was close management and standardization of our materials and documentation to deliver much better timelines. And we recognize the need for rapid delivery, and in the very near future, this will also be further enhanced with the introduction of pre-made off-the-shelf plasmid products like helper and packaging plasmids that are used in viral vector processing.

(00:46:54):
This work also was accompanied by the launch of our dedicated Plasmid DNA Centers of Excellence in the UK. Our Keele site has been around for quite a long time, and it's been producing GMP and non-GMP plasmid products for over 20 years, and it's now our dedicated GMP plasmid manufacturing site undergoing its own capacity expansion.

(00:47:18):
Alderley Park is Charles River's newest CDMO manufacturing site, and it actually opened for business very recently, in October of this year. The site has three fully segregated and independent processing streams, and these can each operate in parallel to deliver over 120 individual batches of high quality plasmid per year. And it's been designed in such a way that the process facility and also the product quality specifications are aligned to principles of GMP.

(00:47:50):
Very briefly, the purpose-built nature of the Alderley Park facility allows us to operate under specific requirements that are necessary for HQ service offering. And importantly, it opens up opportunities to improve the quality of service that we provide to customers but also improve operational efficiencies. It's now open and actively progressing client quality audits, and really it does represent a significant expansion to Charles River's manufacturing capability. And, it's an important step as the company continues to respond to industry feedback and deliver this integrated global network to support advanced therapy products.

(00:48:37):
So to summarize on plasmid grades and their uses, improved guidance is available and it makes the use and manufacture of intermediate grade plasmids much more clear. I think it's important that developers and manufacturers work together to verify the use of appropriate GMP principles for plasmids used in different applications. And what is still clear is that there is no one-size-fits-all approach, but by planning in advance, there are options available that can help minimize later stage disruption and avoid wider program delays.

(00:49:17):
So to summarize overall, when we consider sourcing of plasmid DNA, it is clear that phase-appropriate supply has the ability to support rapid development of a range of advanced therapies. And it does this by reducing costs and timelines. However, there is a clear requirement to minimize quality and cross-contamination impacts that can cause major issues.

(00:49:47):
I hope the presentation has highlighted some of these technical challenges and the need for careful management in some cases, and that allows us as a manufacturer to deliver and also as a consumer to source more consistent products. I encourage all developers to think well ahead and plan for your initial supply by laying a solid foundation through reliable cell banking, and then also consider options like HQ grade plasmid that can allow for an easy transition to GMP grade when the time comes.

(00:50:22):
I hope you found it useful. Again, I wanted to highlight some common challenges and pitfalls that we commonly encounter, but also highlight some of the options that can be incorporated into development and clinical programs to help future-proof supply and ultimately deliver these amazing and life-changing medicines to patients. Thank you. Any questions?

Sarah Hiddleston (00:50:52):
Thank you very much, Andrew, for that presentation. It is now time for our question and answer session. To ask Andrew a question, please type it in where it says, "Type your questions here" and then press submit.

(00:51:09):
I'm going to begin by asking you the questions that I have already on quality standards actually, and I'd like to start with this. Do you expect that there will always be a requirement for intermediate grade plasmid DNA or is it likely that everything's going to move to GMP?

Andrew Frazer (00:51:31):
Yeah, so that's a good question and I guess it's one that's on everybody's mind who's in this business. So there is definitely a trend with manufacturers tightening up on quality control, and in particular testing requirements and validation of methods. So I think if this continues the way it has been recently, it'd be fair to say that HQ would be phased out and replaced completely by GMP plasmid at some point.

(00:51:58):
But equally, we're now receiving much better guidance on where and when we can use HQ grade plasmids, and it does present an opportunity for developers to take these forward for later stage applications. And in fact, we've recently encountered cell therapy programs where HQ grade plasmids planned all the way through to commercial manufacturer. So, if we see continued adoption and acceptance of these types of approaches, I would imagine that intermediate grade plasmids like HQ will be around for the longer term.

Sarah Hiddleston (00:52:37):
Okay. Okay, that's great. Thank you very much. And then on the same subject, there's a trend out there for increased quality standards. Do you expect that those testing specifications are going to follow a similar trend? So will specifications which are acceptable today become unacceptable later further down the line?

Andrew Frazer (00:53:02):
Yeah, again, it's that same worry that would be on everybody's mind who sourcing plasmids. I can definitely, as I said, I can see the level of qualification used for various methods increasing and becoming more aligned to what we see for GMP products. And this goes hand in hand with that risk-based approach for the justification of principles of GMP rather than full GMP for particular applications.

(00:53:31):
I think specifications are a little bit trickier to predict. You can see ... and anybody, I would encourage them to go and read that BioPhorum article. You can see that there is variation in the target specifications that are offered from different manufacturers. And even things, like established specifications, like percentage supercoiled are difficult to standardize and dictate. So I think as we move through and advanced therapies become more well-defined and standardized, then I would say that the specifications around starting materials like plasmid will also become more well-defined.

Sarah Hiddleston (00:54:19):
Okay, thank you very much. Following on from that, what would you say about the differences between regulators between countries? And there's a specific question that's come in about the difference between European and US regulators, but you might be even looking further afield than that. Are there significant differences and are you planning for significant differences?

Andrew Frazer (00:54:43):
Yeah, so we do supply plasmid worldwide, so there are inherent differences between different regulatory bodies. But mainly between the US and the EU, the FDA haven't produced any similar guidance document to the one that's produced by the EMA around principals of GMP, so it's hard to make a direct comparison. But, the FDA did hold a virtual town hall meeting recently where they were asked some very direct questions around the subject, and the response that they gave was very similar to the guidance that was provided by the EMA. And again, they said plasmid used as a starting material didn't have to be manufactured under strict GMP or treated like a direct injectable plasmid product. So I would say in that regard, the guidance is relatively well aligned between the US and the EU.

Sarah Hiddleston (00:55:46):
Great, thank you very much. A quick reminder to our live audience, you do still have time to ask a question. To do so, please just type it in where it says, "Type your questions here" and then press submit.

(00:56:00):
Okay, so maybe on the early stage then, when would you say is the best time to engage with specialist plasmid manufacturers, especially since cost can be quite prohibitive during the early development?

Andrew Frazer (00:56:16):
Yeah, so I think certainly coming from an academic or R&D environment, some of the costs associated with higher quality grade plasmids, I fully appreciate that it can be prohibitive and there are many factors that influence plasmid supply options. But I think as we discussed in the presentation, it is important to at least try to understand what's on offer and plan ahead, and probably the best way to do this is to go out and speak to potential suppliers and weigh up pros and cons.

(00:56:57):
I think my advice would be engage as early as possible, not because we want to attract lots of new business and capture lots of new customers, but what we can do is help support people and help to review how your plasmid supply solution would look in the long term, assess whether the information that you're generating and receiving from your current supplier allows you to easily transition when the time comes to upgrade your quality standards or the supply amounts.

Sarah Hiddleston (00:57:36):
Great, thank you so much. And on supply amounts, you said that your DNA production capability is 120, I think, batches a year. So what is the quantity of the DNA per batch is the question.

Andrew Frazer (00:57:54):
Yeah, so this is related to the Alderley Park site. Alderley Park generates high quality grade DNA currently, so the initial process set up in there is generation of batch sizes at between three and 500 milligrams. And early next year, we'll also be bringing in a process scale up initiative which will deliver one to one and a half grams per batch, and that'll come in within Q1 next year.

Sarah Hiddleston (00:58:30):
Thank you. So manufacturing production scales are quite small compared to, for example, monoclonal antibodies production. Why do you think that is?

Andrew Frazer (00:58:45):
I think it's true, particularly for CDMOs, and some of this I guess is related to what we touched on earlier and the widespread adoption of single-use technology and manufacturing. We talked about some of the advantages of single-use processing streams in the presentation, but I guess the disadvantage is that there is a definite technology limit and that impacts the scale. And, I think the largest commercially available single-use power reactors that people can get their hands on at the moment are in the range of 300 to 500 liters. It works well as it provides flexibility for smaller supply amounts and products with relatively small patient numbers. But, when we look at products with large patient populations like DNA vaccines, we would be looking at tens of kilos per year and above, and this is where we generally see the swap over to dedicated in-house manufacturing with large pharma organizations. At that point, it typically moves to more traditional stainless steel facilities with much larger production skills.

Sarah Hiddleston (01:00:09):
Okay, Andrew, thank you. That is where we are going to need to finish for today. Thank you very much for being with us and for answering our questions, Dr. Andrew Frazer. Thank you very much. I would also like to thank Charles River, our webcast sponsor, and of course, you, the audience for taking the time to be with us today.

(01:00:32):
Do remember, you can watch this again at any time on demand at nature.com/webcasts. Thanks for watching and I hope you'll join us again soon.