This blog post includes verbatim quotes from the presentation by Patricia Pereira Aguilar, Senior Scientist at acib. In addition, an AUDIO discussion ⬇️ of the presentation using ai generation is provided that discusses Patricia Pereira Aguilar's live presentation.
Quotes from the presentation by Patricia Pereira Aguilar, Senior Scientist at acib:
"Salt-tolerant nucleases like M-SAN from ArcticZymes have proven more effective, especially when used in combination with chromatography. Salt disrupts chromatin structure, allowing M-SAN to digest it efficiently." - Patricia Pereira Aguilar (live presentation)
"M-SAN has been shown to significantly improve yields and purity compared to Benzonase, particularly for complex impurities like chromatin." - Patricia Pereira Aguilar (live presentation)
"Salt-tolerant nucleases like M-SAN are critical for efficient removal of chromatin during virus and VLP purification. They improve product safety, yield, and regulatory compliance." - Patricia Pereira Aguilar (live presentation)
"Traditional nucleases are ineffective at digesting chromatin." - Patricia Pereira Aguilar (live presentation)
Audio discussion of the live presentation by Patricia Pereira Aguilar, Senior Scientist at acib, on Jan 28th 2025. This discussion uses ai generation to discuss the presentation based solely on the Patricia Pereira Aguilar's live presentation.
Full transcript below 👇
DNA Removal in Virus and Virus-like particle Downstream Processing
[00:00:00] Hey, did you hear Patricia Aguilar's ACIB presentation on virus and VLP purification, tackling chromatin impurities with salt tolerant nucleases? It's packed with fascinating insights into the challenges of purifying viruses and VLPs, especially the often overlooked problem of chromatin contamination.
Indeed, her presentation really highlights the need for more effective strategies to address this persistent impurity, particularly as we move towards large scale production of viral vectors and vaccines. Absolutely, and she presented some very compelling data on the potential of salt tolerant nucleases like M-SAN to revolutionize this process.
Yes, M-SAN's ability to function effectively in salt environments where chromatin structure is disrupted offers a significant advantage over traditional nucleases. Before we delve into the specifics of M-SAN, let's quickly recap the basics of viruses and VLPs for context. Certainly, viruses are those microscopic, infective agents composed of genetic material encased in a protein shell, while VLPs are essentially virus mimics, structurally [00:01:00] similar but lacking the infectious genetic core.
And they vary considerably in size, right? Aguilar mentioned viruses ranging from tiny AAVs to those resembling microscopic worms. Precisely. The diversity in size and structure presents a significant challenge for purification, as separation techniques must be tailored to each specific virus or VLP. And we can't forget the distinction between enveloped and non enveloped viruses.
Indeed. Enveloped viruses acquire a lipid membrane from the host's cell during their exit, adding another layer of complexity to the purification process. So how are these viruses and VLPs produced in the lab? Virus production typically involves infecting cell cultures with a viral stalk, prompting the cells to become virus factories, releasing either enveloped or non enveloped viruses.
VLP production bypasses the viral genome entirely. Instead, we introduce the genetic instructions for the viral proteins into host cells, allowing these proteins to self assemble into VLPs. Now, shifting our focus to [00:02:00] applications, VLPs seem to hold immense potential in various fields. Absolutely. Their versatility in presenting antigens makes them ideal for vaccine development, both prophylactic and therapeutic.
And they're being explored for gene therapy, antiviral treatments, and even targeted cancer therapies. Precisely. Their ability to encapsulate therapeutic agents and target specific cells makes them highly attractive for a wide range of applications. However, Aguilar emphasized that purifying viruses in VLPs is far from straightforward.
Indeed, numerous impurities originating from the production process pose a significant challenge. Impurities like cell debris, host DNA, Proteins and even exosomes, those tiny vesicles secreted by cells. Correct. And removing these impurities is crucial not only for product safety, but also to ensure regulatory compliance.
Aguilar specifically highlighted the importance of removing host cell DNA, especially in vaccine development. Absolutely. Residual DNA can trigger unwanted immune responses and faces strict regulatory limits, [00:03:00] particularly for vaccines where levels must be below 10 nanograms per dose. And this is where the problem of chromatin contamination takes center stage.
Indeed, chromatin composed of DNA tightly wound around histone proteins presents a persistent and often underestimated challenge. Its compact structure makes it resistant to traditional purification methods, and its size range overlaps with that of viruses and VLPs making separation difficult.
Precisely. Traditional nucleases like Benzonase often struggle to effectively digest chromatin due to its tightly packed configuration. So how does M-SAN address this challenge? M-SAN exhibits remarkable salt tolerance, allowing it to function efficiently in high salt conditions where chromatin structure becomes more relaxed and accessible.
This is where I think the real power of M-SAN lies, its ability to not only remove DNA, but to do so efficiently, potentially simplifying purification workflows and reducing production costs.
That's a crucial point, especially as we move towards large scale production of viral vectors and vaccines for [00:04:00] a wider range of applications. Absolutely. And this brings us to the second study highlighted by Aguilar conducted by Mayer et al. Focusing on measles virus . They used western blot , to visualize the presence of chromatin, right?
Correct. Western blots targeted histone H3, a marker for chromatin,. And what did their findings reveal? Their results were quite striking. Even after treatment with traditional nucleases, chromatin persisted in the samples clearly visible and detectable by Western blot.
However, when they used M-SAN the chromatin was effectively eliminated, becoming undetectable by Western blot. Precisely. Their findings underscore the limitations of traditional nucleases in tackling chromatin contamination and highlight the superior performance of M-SAN in this regard.
So M-SAN seems to offer a promising solution to this persistent challenge, but as with any new technology, it's important to consider its broader implications and potential impact on the field. Absolutely, and that's where I think Aguilar's presentation really shines. She doesn't simply present M-SAN as a silver bullet, but [00:05:00] encourages us to think critically about its integration into existing purification workflows and its potential long term effects.
In other words, we need to carefully evaluate not only its immediate benefits, but also its potential impact on downstream processes and product quality. Precisely. And this leads us to another crucial aspect of purification, the role of chromatography. Aguilar mentioned two specific types, flow through chromatography and affinity chromatography.
Yes, flow through chromatography acts like a sieve, retaining smaller impurities while allowing larger viruses or VLPs to pass through, while affinity chromatography exploits specific interactions for more targeted separation. So how does M-SAN's ability to remove chromatin potentially enhance the effectiveness of these chromatographic techniques?
By eliminating chromatin early on, M-SAN can prevent it from interfering with the binding and elution processes in chromatography, leading to improved resolution and purity. That makes sense. And I imagine this also translates into increased efficiency and reduced production costs. [00:06:00] Indeed. And this is where I think the true potential of M-SAN lies, its ability to not only improve product safety, but also to streamline the entire purification process, making it more efficient and cost effective.
So M-SAN seems to hold immense promise for the future of virus and VLP production. But as with any new technology, there's still much to learn and explore. Absolutely, and that's what makes this field so exciting. We're constantly pushing the boundaries of what's possible, and tools like M-SAN are paving the way for a new era of biopharmaceutical manufacturing.
Now let's transition to a deeper exploration of the benefits and challenges of M-SAN. as well as its potential long term impact on the field. Building upon our previous discussion of M-SAN's capabilities, let's delve deeper into the potential benefits it offers for virus and VLP production. That's an excellent segue.
We've already touched on M-SAN's potential to improve product safety, but I'd like to explore this further. How exactly does its ability to remove chromatin translate to enhance safety for vaccines and therapeutics? [00:07:00] Chromatin, as we discussed, can elicit unwanted immune responses. In the context of vaccines, this can lead to inflammation and potentially even autoimmune reactions.
By effectively removing chromatin, M-SAN minimizes this risk, ensuring that the immune response is directed towards the intended target antigen and not contaminating DNA. That's a crucial point, particularly for sensitive applications like gene therapy, where minimizing off target effects is Paramount.
Precisely. And it's not just about safety. M-SAN also has the potential to significantly increase yields, a critical factor in the economic viability of biopharmaceutical production. Can you elaborate on that? How does removing chromatin contribute to higher yields? Chromatin's complex structure and size can impede various downstream purification steps.
It can clog filters, reduce the efficiency of chromatography columns, . So it's essentially a bottleneck in the manufacturing process. A very apt analogy. By removing chromatin early on, M-SAN streamlines the entire workflow leading to less product [00:08:00] loss and ultimately higher yields.
This increased efficiency naturally translates into cost savings, which is particularly relevant for complex and expensive therapies like gene therapies. Absolutely. Fewer purification steps, less product loss, and faster processing times all contribute to reduced manufacturing costs, making these life saving treatments more accessible to patients in need.
And let's not forget the regulatory aspect. We know that regulatory agencies have strict limits on DNA contamination in biopharmaceuticals. Indeed, meeting these stringent requirements can be challenging with traditional methods. M-SAN's ability to consistently reduce DNA levels below the regulatory thresholds simplifies the approval process and mitigates the risk of costly delays.
So M-SAN seems to tick all the boxes, enhanced safety, increased yields, and improved regulatory compliance. However, as with any new technology, there are likely challenges and areas for future research. Certainly one area that requires attention is the development of [00:09:00] more robust and sensitive analytical tools for detecting and quantifying chromatin.
Why is this so crucial? Current methods for chromatin quantification often rely on indirect measurements, such as western blot detection of histone proteins. While useful, these techniques don't directly measure the DNA content within chromatin, limiting our ability to accurately assess its presence. Developing new analytical methods that can directly quantify chromatin's DNA content will be essential for establishing robust quality control measures and optimzing M-SAN's implementation in various purification workflows. Another area that warrants further investigation is the biological impact of residual chromatin.
While we know it can trigger immune responses, the full extent of its potential effects remains unclear. That's correct. More research is needed to determine how different levels of residual chromatin might influence the safety and efficacy of vaccines and gene therapies. For instance, could there be a threshold level below which chromatin poses no significant risk?
Or could [00:10:00] even trace amounts have unforeseen long term consequences? These are critical questions that need to be addressed through rigorous scientific investigation. moving beyond simply detecting chromatin to understanding its biological impact in different contexts will be essential for harnessing its potential benefits while mitigating any potential risks.
Now, having explored the potential benefits and challenges associated with M-SAN, let's shift our focus to its real world applications and examine how it's being integrated into biopharmaceutical manufacturing processes. In this final segment, we'll shift our focus from the theoretical to the practical, exploring how M-SAN is being implemented in real world biopharmaceutical production.
Indeed, let's examine how this innovative tool is shaping the manufacturing landscape for vaccines, gene therapies, and other biotherapeutics. Let's start with gene therapy, a field that holds immense promise for treating a wide range of genetic disorders. We've discussed the importance of viral vectors as delivery vehicles for therapeutic genes, but their production comes with inherent [00:11:00] purification challenges.
Absolutely, and M-SAN is proving to be a valuable asset in addressing these challenges. Its effectiveness in removing chromatin contamination is particularly relevant for lentiviral vectors, which are often sensitive to the high salt concentrations traditionally used to disrupt chromatin structure. So, M-SAN offers a gentler alternative preserving the integrity of these sensitive vectors.
Precisely, by enabling efficient chromatin digestion at physiological salt concentrations, M-SAN not only improves the purity , but also increases their overall yield, a crucial factor in the economic viability of these often expensive therapies.
It's also being utilized in the production of AAVs, another prominent player in the gene therapy arena. Yes, AAVs are gaining increasing traction, particularly in the burgeoning field of gene editing. Their ability to deliver gene editing tools like CRISPR Cas9 has opened up exciting new possibilities for treating genetic diseases at their root.
And M-SAN plays a critical role in ensuring the safety and efficacy of these AAV based therapies. [00:12:00] therapies by effectively removing contaminating chromatin. Indeed, the purity of viral vectors is paramount, especially when we're talking about manipulating the human genome. M-SAN's ability to contribute to this high level of purity makes it an invaluable tool in the advancement of gene therapy.
Well, this brings our deep dive into the world of virus and VLP purification to a close. We've journeyed from the fundamentals of these tiny biological entities to the complexities of chromatin contamination and the transformative potential of salt tolerant nucleases like M-SAN. It's been a pleasure delving into these topics with you, exploring the challenges, the innovations, and the exciting possibilities that lie ahead in this rapidly evolving field. We hope this deep dive has provided you with valuable insights and perhaps even sparked your own curiosity about the intricate world of biopharmaceutical development. As we continue to push the boundaries of scientific understanding, one thing remains certain. The future of biotherapeutics is fueled by innovation, [00:13:00] collaboration, and a relentless pursuit of improving human health.
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