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class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-2404 post type-post status-publish format-standard has-post-thumbnail category-connect-to-science category-covid-19-research category-general entry" aria-label="A Cinderella Story: How a Once-Challenged Method of Protein Expression Became an Indispensable Tool in the COVID-19 Response"><header class="entry-header"><h1 class="entry-title">A Cinderella Story: How a Once-Challenged Method of Protein Expression Became an Indispensable Tool in the COVID-19 Response</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"> 01.11.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="aligncenter size-full wp-image-2483" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/Discover_Expi293.jpg" alt="Illustration of an antibody attacking a coronavirus " width="1000" height="682" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/Discover_Expi293.jpg 1000w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/Discover_Expi293-300x205.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/Discover_Expi293-768x524.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/Discover_Expi293-96x65.jpg 96w" sizes="(max-width: 1000px) 100vw, 1000px" /></p> <p> </p> <p><em>by Lisa Kepler </em></p> <p>Once upon a time there was a novel virus that had seized the globe like a clenched hand squeezing tighter and tighter. Hospitals were the first to crack under the immense pressure –overflowing with infected patients, running ventilators and makeshift beds from tents in the parking lot. The death toll was rising, and a world frantically looking for ways to slow the rapid spread of the highly infectious coronavirus SARS-CoV-2.</p> <p>It was March 2020, just one week after the US national laboratories had shut down alongside much of the country. Staff were instructed to go home and shelter in place. But no sooner had he settled in at home when Dr. Dominic Esposito, director of the Protein Expression Laboratory at the Frederick National Laboratory for Cancer Research, got a call. It was from the National Institutes of Health (NIH), and it was urgent. They needed him back.</p> <p>To understand why Esposito – not a virologist but a protein expert – came to be one of the key players in developing the world’s first COVID tests, vaccinations, and treatments, and how decades of basic research innovation can set the stage for nimble response to an urgent crisis, we need to start with a different story. We need to start at the molecular scale, with viruses, cells, and proteins.</p> <p> </p> <h2><strong>Breaking the ‘express, fail, repeat’ cycle of early development</strong></h2> <p>Unlike bacterial pathogens, viruses are not living things. They are, quite simply, two parts of an extremely small whole: nucleic acid in a mantle of protein, arranged so simply that these microbes need a host to reproduce but intricately enough to bring an entire planet to a halt.</p> <p>The success of early drug and vaccine discovery also comes down to just two ingredients: a promising therapeutic candidate molecule and a suite of reliable disease targets. The candidates get a lot of attention in the public sphere because they can play the exciting role of main character in a complex chemical interaction that claims a vast supporting cast. What new molecular combination might be the next blockbuster to change a therapeutic landscape? But bench scientists also need to create and study a high volume of reliable targets. These targets are the many proteins in cells that are involved in a disease process, which therapeutics can aim to interrupt. To properly understand drug and vaccine mechanics, researchers need samples of those relevant proteins. And a lot of them.</p> <p>Prior to 2012, the only way to achieve high-yield protein expression – the process of optimizing cells like living factories to generate as much protein as possible – involved integrating a foreign gene into the host cell’s genome to develop something called a stable cell line. A stable line gives rise to identical populations that can express a desired protein in a consistent, heritable way. Creating these lines is a foundational process in large scale drug development where targets are known factors, but in early discovery the sheer time and labor required to keep generations of cells replicating can really bog things down. A lab might go through dozens of experimental iterations before finding the right protein target, and the need to re-establish stable lines for each protein means every ‘trial-and-error’ is a weeks- or months-long affair and a tremendous drag on efficient innovation. In the meantime, patients are waiting, and drug developers are burning cash.</p> <p> </p> <h2><strong>A new era for protein expression</strong></h2> <p>Stable expression presents a significant roadblock to rapid therapeutic developments, but there have always been other roads to protein production. Transient expression, for example, is a temporary period of protein production triggered by a foreign gene that is not integrated in a heritable way into the host cell genome. Transient lines are the pop-up shop to the stable line’s established factory line. It seems like an obvious choice, so why not use transient expression?</p> <p>While transient cell lines do offer a faster, less expensive way to generate proteins of interest, they can have a fatal flaw: historically, these lines haven’t been able to yield enough protein for validating drug targets. For that reason, they were generally overlooked in R&D. Still, Dr. Henry Chiou, general manager in Delivery and Protein Expression at Thermo Fisher Scientific, saw promise.</p> <p>“We knew that if we could optimize the transient expression process, we could get much more protein in a small culture volume,” said Chiou. “To achieve that, we took the guesswork out of the process by creating a kit that included everything: a high-expressing HEK293 cell line, a chemically defined serum–free culture medium, and a high-efficiency transfection reagent with specialized enhancers. With each component optimized, achieving high-yield expression every time got a lot easier.”</p> <p><iframe title="Expi293™ Expression System - Meet the inventor series" width="760" height="428" src="https://www.youtube.com/embed/O_nej2BHJEg?start=68&feature=oembed" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe></p> <p>Chiou’s kit worked beautifully. Dubbed the <a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expi293-expression-system.html">Gibco™ Expi293™ Transient Expression System</a> at debut, the novel expression platform offered up to six-times higher yield than previous transient systems and enabled protein harvest in as little as one week, which was weeks faster than that achievable via stable development. Because the Expi293 system enabled quick cell recovery, labs could produce sufficient amounts of many candidate proteins in parallel for testing. Episodes of trial and error were still integral to discovery, but the Expi293 system allowed researchers to recover from “failures” much more quickly and find potential successes faster.</p> <p>Suddenly, the research community began to open its eyes to the greater promise of transient protein expression—and the Expi293 system was the belle of the ball.</p> <h2><strong>Science versus the spike</strong></h2> <p>By the time COVID-19 became part of our daily vernacular, the Expi293 system had already been the go-to choice across the industry for transient expression in mammalian 293 cells for nearly a decade. But it was about to meet its most daunting test yet. No one could have prepared the world for the challenges of the global pandemic driven by a virus that was as complex to study as it was unrelenting—and this brings us back to that fateful phone call between the NIH and Dr. Esposito.</p> <p>“As they worked to develop COVID-19 vaccines, the NIH began to realize that the scientific community was stumped on a major obstacle: replicating COVID’s spike protein,” said Esposito.</p> <div id="attachment_2484" style="width: 310px" class="wp-caption alignright"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-2484" class="wp-image-2484 size-medium" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/expi-300x277.jpg" alt="Gibco™ Expi293™ Expression System Kit" width="300" height="277" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/expi-300x277.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/expi-1024x945.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/expi-768x709.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/expi.jpg 1452w" sizes="(max-width: 300px) 100vw, 300px" /><p id="caption-attachment-2484" class="wp-caption-text">Gibco™ Expi293™ Expression System Kit</p></div> <p>Even if you don’t <em>think</em> you know the spike protein, you probably do. It’s the star of most of the up-close photos you’ve seen of SARS-CoV-2 virus particles—the proteins that jut out from the surface like a stellar corona. (Incidentally, it’s also responsible for the virus’s moniker; “coronavirus” is rooted in the Latin word for crown.) These spikes are the infection pathway for the virus, allowing it to recognize and bind to human proteins on the surface of cells. That’s why it was the ideal target for vaccines—both the Moderna and Pfizer/BioNTech vaccines target the spike.</p> <p>“On our call, NIH had two urgent asks,” said Dr. Esposito. “First, they needed us to create spike protein for serology assay antigens to understand how much of the U.S. population was affected by COVID-19. Second, they needed us to come up with a way to create enough spike protein for Moderna’s phase II vaccine trial analysis. It was a time when the whole world wanted to find ways to help, and my team was no different; we couldn’t get to work fast enough.”</p> <p>As it turned out, the Expi293 system would be critical to delivering on both requests. But it wouldn’t be easy. Given the ubiquitous presence of related coronaviruses in the population (including the common cold), Dr. Esposito’s team needed to produce assays that were highly specific to the SARS-CoV-2 spike protein. At the outset, he encountered the same challenge that research teams across the globe were encountering: if he produced large yields of the spike protein, they were not consistent; if he produced consistent yields, they weren’t large enough.</p> <p>Undeterred, Esposito’s team continued to try different formulations over a compressed period of time, which was possible because of the rapid cell recovery enabled by the Expi293 system. They changed how they grew the cells, the timing of collecting the protein, temperature control, and nearly every other variable they could think of. Using the Expi293 system, they finally found a solution that made five milligrams of spike protein for every liter of expression culture—a significant improvement from what the scientific community had achieved to date. What’s more, the spike proteins were stable and didn’t fall apart easily. That meant they not only worked well in tests to check for COVID-19 antibodies, but they also successfully supported Moderna in its phase II vaccine studies.</p> <p>Esposito <a href="https://pubmed.ncbi.nlm.nih.gov/32504802/">documented his team’s work</a> in the October 2020 issue of <em>Protein Expression Purification</em>, where it became a highly cited go-to resource in COVID-19 science.<sup> 1 </sup>Since then, Esposito and his team have made the spike for every variant of concern that has been identified. Their work paved the way for not only the COVID-19 vaccines, but also more accurate and affordable COVID-19 antibody tests and monoclonal antibody therapies for immunocompromised patients.<sup>2-4 </sup></p> <p>“There is no way we could have created the high volumes of spike protein needed without the Expi293 system,” said Esposito. “When it comes quickly generating high protein yields with low volume, the Expi kits are exceptional—and in a pandemic environment when answers couldn’t wait, achieving this kind of speed and quality was everything.”</p> <div style="width: 760px;" class="wp-video"> <video class="wp-video-shortcode" id="video-2404-1" width="760" height="428" preload="metadata" controls="controls"><source type="video/mp4" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/PG2344-PJT7096-COL25708-SB-293-Cell-Handling-Technical-Video-Global-F-1.mp4?_=1" /><a href="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/PG2344-PJT7096-COL25708-SB-293-Cell-Handling-Technical-Video-Global-F-1.mp4">https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/01/PG2344-PJT7096-COL25708-SB-293-Cell-Handling-Technical-Video-Global-F-1.mp4</a></video></div> <h2></h2> <h2></h2> <h2><strong>The evolution of Expi systems</strong></h2> <p>In Cinderella, the Fairy Godmother says, “If you’d lost all your faith, I wouldn’t be here…and I am!” In truth, the same could be said for every scientific discovery, be it COVID vaccines, cancer breakthroughs or cell therapies.</p> <p>As scientists, we are curious by nature, constantly pushing the boundaries of discovery to see what we can learn next. That’s why the innovation didn’t stop with the Expi293 system’s introduction back in 2012. Following that, its developers seized on the opportunity to create an optimized system approach for CHO cells in 2015 via the <a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expicho-expression-system.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt10933_blg50609_0so_blg_il_awa_og_s00_C2SExpiBlogCHO">Gibco™ ExpiCHO™ Expression System</a>.</p> <p>ExpiCHO is tailored for transient protein expression in Chinese hamster ovary (CHO) cells which are used to manufacture 70% of today’s biologics. This system moved the needle in protein yields from milligrams to grams per liter, a massive improvement in productivity. The next optimized systems launched in 2018 with the first chemically-defined baculovirus expression system, the <a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/insect-protein-expression/expisf-expression-system.html?cid=bid_clb_pex_r01_co_cp1533_pjt10065_gbpex2765_0db_ccd_nd_tls_og_s00_CCDexpertsession">Gibco™ ExpiSf™ Expression System</a>. Within each iteration of the system, Gibco crafted a complete, optimized system that had the ideal components to suit each cell line. Optimizing the Expi293 system also opened the door later to the development of optimized <a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/lv-production-workflow/lentivirus-production-research.html">LV-MAX Lentiviral</a> and <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">AAV-MAX</a> production systems for <a href="https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection.html">viral transfection</a> in cell and gene therapy.</p> <p>The Expi293 expression system took the concept of a truly optimized system of cells, media, and transfection reagents and built on it to fuel a new generation of scientific discoveries. As for what discoveries and technologies will come next in 2024? We’ll just have to wait and see.</p> <p> </p> <h3><strong>» Learn more about rapid, high-yield transient HEK 293 expression with </strong><a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expi293-expression-system.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt10933_blg50609_0so_blg_il_awa_og_s00_C2SExpiBlogExpi293"><strong>the Expi293 system</strong></a><strong><br /> </strong></h3> <p><em>Want to read more stories like this? </em><a href="https://www.thermofisher.com/us/en/home/global/forms/connect-science-subscribe.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt10933_blg50592_0so_blg_il_awa_og_s00_C2SOncoBlogC2SSub"><em>Subscribe to Connect to Science</em></a><em>, your portal for life science news.</em></p> <p><strong> </strong></p> <p>###</p> <p> </p> <h3><strong>References</strong></h3> <ol> <li>Esposito D, Mehalko J, Drew M, Snead K, Wall V, Taylor T, Frank P, Denson JP, Hong M, Gulten G, Sadtler K, Messing S, Gillette W. Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays. Protein Expr Purif. 2020 Oct;174:105686. doi: 10.1016/j.pep.2020.105686.</li> <li>Villarraza J, Fuselli A, Gugliotta A, Garay E, Rodríguez MC, Fontana D, Antuña S, Gastaldi V, Battagliotti JM, Tardivo MB, Alvarez D, Castro E, Cassataro J, Ceaglio N, Prieto C. A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes. Appl Microbiol Biotechnol. 2023 Jun;107(11):3429-3441. doi: 10.1007/s00253-023-12520-5. Epub 2023 Apr 24. PMID: 37093307; PMCID: PMC10124706.</li> <li>Jaki L, Weigang S, Kern L, Kramme S, Wrobel AG, Grawitz AB, Nawrath P, Martin SR, Dähne T, Beer J, Disch M, Kolb P, Gutbrod L, Reuter S, Warnatz K, Schwemmle M, Gamblin SJ, Neumann-Haefelin E, Schnepf D, Welte T, Kochs G, Huzly D, Panning M, Fuchs J. Total escape of SARS-CoV-2 from dual monoclonal antibody therapy in an immunocompromised patient. Nat Commun. 2023 Apr 10;14(1):1999. doi: 10.1038/s41467-023-37591-w. PMID: 37037847; PMCID: PMC10085998.</li> <li>Alvim RGF, Lima TM, Rodrigues DAS, Marsili FF, Bozza VBT, Higa LM, Monteiro FL, Abreu DPB, Leitão IC, Carvalho RS, Galliez RM, Castineiras TMPP, Travassos LH, Nobrega A, Tanuri A, Ferreira OC Jr, Vale AM, Castilho LR. From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics. Biochem Eng J. 2022 Aug;186:108537. doi: 10.1016/j.bej.2022.108537. Epub 2022 Jul 16. PMID: 35874089; PMCID: PMC9287463.</li> </ol> <p> </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 specified.<br /> </em><em>For Research Use Only. 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