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class="breadcrumb-current-title">Addressing the Plasmid Production Bottleneck: Automated Maxi-Scale Purification</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-3119 post type-post status-publish format-standard has-post-thumbnail category-connect-to-science category-general entry" aria-label="Addressing the Plasmid Production Bottleneck: Automated Maxi-Scale Purification"><header class="entry-header"><h1 class="entry-title">Addressing the Plasmid Production Bottleneck: Automated Maxi-Scale Purification</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/lifeinthelabstaff/" title="Posts by Life in the Lab Staff" rel="author">Life in the Lab Staff</a> </span> <span class="post-info-date"> 07.02.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="aligncenter size-full wp-image-3124" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/image-2.jpg" alt="PlasmidPro" width="1100" height="700" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/image-2.jpg 1100w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/image-2-300x191.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/image-2-1024x652.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/06/image-2-768x489.jpg 768w" sizes="(max-width: 1100px) 100vw, 1100px" /></p> <h2><em>How automated plasmid production can help viral vectors realize their true potential in gene therapy and beyond</em></h2> <p> </p> <p>Viral vectors have exhibited profound clinical promise and have gained significant attention as they show more and more success in certain applications. Of these viral vectors, <a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/aav-production-workflow/adeno-associated-virus-production-cell-gene-therapy.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SAAVProdWorkflow">Adeno-associated virus (AAV)-based vectors</a> and <a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/lv-production-workflow.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SLVProdWorkflow">lentiviral vectors (LVV)</a> represent the 2 primary vector types utilized in gene therapy. While both vector types are extensively used in the clinic, AAV is typically the primary method for <em>in vivo </em>delivery and ideal for the replacement of nonfunctional genes and modulating disease progression through gene addition. Alternatively, LVVs are often utilized for <em>ex vivo </em>gene correction and as a result, are the preferred method for the treatment of monogenic diseases and adoptive cell therapies.</p> <p>Given this rapid clinical progress in the viral vector space with the success of AAV and LVV based applications, manufacturers and researchers are placing a heightened emphasis on scale-up technologies that can further enhance supply of these vectors as the demand continues to grow. However, a significant bottleneck has formed around this production of viral vectors. Therefore, the key question poses itself into how can we leverage innovation and technology advancements to overcome this current bottleneck and scale up the manufacture of critical raw materials needed for gene therapies?</p> <h3><strong>Plasmid Use in Viral Vector Production </strong></h3> <p>As mentioned above, this rapid clinical progress has emphasized the need for scale-up technologies that can support the supply of these vectors as the demand continues to grow. Both recombinant AAV and LVV are produced using transient transfection of <a href="https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/plasmid-isolation.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SPlasmidIso">plasmid DNA (pDNA)</a> containing the required genetic instructions to produce the vector in cell culture.<sup>16</sup> As a result, the demand for pDNA has skyrocketed over the past few years and now represents a significant bottleneck from a supply and consistency perspective.<sup>17 </sup>We spoke to the thought leaders at Thermo Fisher around specific pain points in pDNA production and innovations currently ongoing to alleviate this pDNA bottleneck for viral vector manufacturing. “Consistency and getting the same results every time in pDNA is crucial for the success” said Jenny Clark, a Product Manager at Thermo Fisher. Contaminants commonly present in pDNA preparations include host <em>E. coli</em>-derived genomic DNA, endotoxins, proteins and RNAs. Further, alternative, less functional conformations of the pDNA itself, such as open-circle (nicked) and linear forms produced via enzymatic or shear-induced damage to supercoiled plasmid DNA can also be problematic.<sup>17,18</sup> Cumulatively, these pDNA contaminants can adversely affect the downstream cell culture performance, resulting in reduced infectious virus titer as well as compromised vector preparations.</p> <h3><strong>Current Strategies and Pitfalls in Downstream Plasmid Preparation </strong></h3> <p>While Clark noted the challenge with consistency of pDNA preps using manual methods, this poses a major hurdle for current pDNA manufacturing methods where large amounts of high-quality, consistent DNA is required. One of the most popular methods is performed using a manual and open process that requires extensive hands-on time.<sup>19 </sup>Though this manual process may initially seem cost effective, in reality, it introduces additional resource requirements through lost time from repetitive centrifugation steps and the demand for highly trained manpower to complete pDNA purification. “Typically, you’re looking at a minimum of 2 hours hands on manual time required to complete just one purification,” said Clark. “Plasmid groups require sometimes 10’s if not hundreds of plasmid purifications. Whole teams are dedicated to just plasmid purification,” Clark noted. Further, critical quality attributes of the pDNA preparations can vary based on the operator performing the purification. Given that DNA preparation quality can influence functional performance, additional training and resource expenditure is often required to better harmonize operators in plasmid preparation.<sup>19</sup></p> <h3><strong>Driving Innovation in Genomic Medicine </strong></h3> <p>Thermo Fisher Scientific has recognized this significant resource requirement for pDNA purification as well as the risk for the introduction of pDNA variability using manual methods. “The hardest part in doing this process (manually) is you can’t walk away in the middle of it. You are literally chained to the bench from beginning to end of this process since a lot of it is time dependent,” said Clark. “Automation can excel in (pDNA) quality metrics…supercoiled DNA status and minimal shear, removal of endotoxin, will improve,” said Clark.</p> <p>The <a href="https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/plasmid-isolation/automated-plasmid-purification.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SPlasmidPro">KingFisher PlasmidPro</a> has been developed to produce high-purity and consistent pDNA using a fully automated end-to-end plasmid purification platform. Capable of purifying plasmid from culture volumes up to 150mL with minimal hands-on time in under 75 minutes, the system was designed to free highly trained and valuable resources while removing plasmid prep variability for batch to batch. Together, automation of plasmid production can enhance pDNA quality and availability, breaking the bottleneck in viral vector production and ensuring success in downstream applications.<strong> </strong></p> <h2><strong><a href="https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/plasmid-isolation/automated-plasmid-purification.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SPlasmidPro">» Learn more about maxi-scale automated plasmid production</a> </strong></h2> <p><em>Want to read more stories like this? </em><a href="https://www.thermofisher.com/us/en/home/global/forms/connect-to-science-digital-care-package.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt12448_blg54886_0so_blg_il_awa_og_s00_C2SDigitalCarePkg"><em>Subscribe to Connect to Science</em></a><em>, your portal for life science news.</em></p> <p><strong> </strong><strong> </strong></p> <h4><strong>References</strong></h4> <ol> <li>Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. <em>Nature Reviews Drug Discovery</em> vol. 18 358–378 Preprint at https://doi.org/10.1038/s41573-019-0012-9 (2019).</li> <li>Keeler, A. M. & Flotte, T. R. Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here? <em>Annu Rev Virol</em> <strong>6</strong>, 601–621 (2019).</li> <li>Nathwani, A. C. Gene therapy for hemophilia. 1–8 (2019).</li> <li>Jablonka, S., Hennlein, L. & Sendtner, M. Therapy development for spinal muscular atrophy: perspectives for muscular dystrophies and neurodegenerative disorders. <em>Neurological Research and Practice</em> vol. 4 Preprint at https://doi.org/10.1186/s42466-021-00162-9 (2022).</li> <li>Miniarikova, J., Evers, M. M. & Konstantinova, P. Translation of MicroRNA-Based Huntingtin-Lowering Therapies from Preclinical Studies to the Clinic. <em>Molecular Therapy</em> vol. 26 947–962 Preprint at https://doi.org/10.1016/j.ymthe.2018.02.002 (2018).</li> <li>Ishikawa, K., Weber, T. & Hajjar, R. J. Human cardiac gene therapy. <em>Circulation Research</em> vol. 123 601–613 Preprint at https://doi.org/10.1161/CIRCRESAHA.118.311587 (2018).</li> <li>Nam, Y., Moon, G. J. & Kim, S. R. Therapeutic potential of aav1-rheb(S16h) transduction against neurodegenerative diseases. <em>International Journal of Molecular Sciences</em> vol. 22 1–19 Preprint at https://doi.org/10.3390/ijms22063064 (2021).</li> <li>Santiago-Ortiz, J. L. & Schaffer, D. V. Adeno-associated virus (AAV) vectors in cancer gene therapy. <em>Journal of Controlled Release</em> <strong>240</strong>, 287–301 (2016).</li> <li>Perry, C. & Rayat, A. C. M. E. Lentiviral vector bioprocessing. <em>Viruses</em> <strong>13</strong>, (2021).</li> <li>Gutierrez‐Guerrero, A., Cosset, F. L. & Verhoeyen, E. Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy. <em>Viruses</em> <strong>12</strong>, (2020).</li> <li>Milone, M. C. & O’Doherty, U. Clinical use of lentiviral vectors. <em>Leukemia</em> vol. 32 1529–1541 Preprint at https://doi.org/10.1038/s41375-018-0106-0 (2018).</li> <li>Cavazzana-Calvo, M. <em>et al.</em> Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia. <em>Nature</em> <strong>467</strong>, 318–322 (2010).</li> <li>Eichler, F. <em>et al.</em> Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. <em>New England Journal of Medicine</em> <strong>377</strong>, 1630–1638 (2017).</li> <li>Labbé, R. P., Vessillier, S. & Rafiq, Q. A. Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives. <em>Viruses</em> <strong>13</strong>, 1528 (2021).</li> <li>Rittiner, J., Cumaran, M., Malhotra, S. & Kantor, B. Therapeutic modulation of gene expression in the disease state: Treatment strategies and approaches for the development of next-generation of the epigenetic drugs. <em>Frontiers in Bioengineering and Biotechnology</em> vol. 10 Preprint at https://doi.org/10.3389/fbioe.2022.1035543 (2022).</li> <li>Breckpot, K., Aerts, J. L. & Thielemans, K. Lentiviral vectors for cancer immunotherapy: Transforming infectious particles into therapeutics. <em>Gene Therapy</em> vol. 14 847–862 Preprint at https://doi.org/10.1038/sj.gt.3302947 (2007).</li> <li>Tejeda-Mansir, A. & Montesinos, R. M. <em>Upstream Processing of Plasmid DNA for Vaccine and Gene Therapy Applications</em>. <em>Recent Patents on Biotechnology</em> vol. 2 (2008).</li> <li>Sousa, A., Sousa, F. & Queiroz, J. A. Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification. <em>J Chromatogr B Analyt Technol Biomed Life Sci</em> <strong>879</strong>, 3507–3515 (2011).</li> <li>Cohen, M. <em>et al.</em> A fully automated high-throughput plasmid purification workstation for the generation of mammalian cell expression-quality DNA. <em>SLAS Technol</em> <strong>27</strong>, 227–236 (2022).</li> </ol> <p><strong>###</strong></p> <p><em>© 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise</em> <em>specified.</em></p> <p><em>For Research Use Only. 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