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aria-label="breadcrumb separator">/</span> <span class="breadcrumb-link-wrap"><a class="breadcrumb-link" href="https://www.thermofisher.com/blog/analyteguru/uhplc/"><span class="breadcrumb-link-text-wrap">UHPLC</span></a><meta ></span> <span aria-label="breadcrumb separator">/</span> <span class="breadcrumb-current-title">Size Exclusion Chromatography of Adeno-associated Viruses</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-17856 post type-post status-publish format-standard has-post-thumbnail category-uhplc tag-aav tag-uhplc entry" aria-label="Size Exclusion Chromatography of Adeno-associated Viruses"><header class="entry-header"><h1 class="entry-title">Size Exclusion Chromatography of Adeno-associated Viruses</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/analyteguru/tf-author/mauro-de-pra/" title="Posts by Mauro De Pra" rel="author">Mauro De Pra, HPLC Product Marketing Manager, Thermo Fisher Scientific</a> </span> <span class="post-info-date"> 07.22.2024 </span> </header><div class="entry-content"> <h3 class="wp-block-heading" id="h-why-should-you-consider-sec-to-analyze-aav-aggregates"><strong>Why should you consider SEC to analyze AAV aggregates?</strong></h3> <p>In the field of adeno-associated viruses (AAV) analysis, understanding the critical attribute of aggregation is of utmost importance for therapeutic safety and efficacy.</p> <p>This blog post will highlight why Size Exclusion Chromatography (SEC) columns combined with UHPLC-FLD-UV analysis is the appropriate technique for analyzing your AAV aggregates.</p> <p>Want to keep up with the latest technology in bioseparation? <a href="https://www.thermofisher.com/us/en/home/global/forms/industrial/mdi-column-technology.html" target="_blank" rel="noreferrer noopener">Sign up today</a> and <a href="https://event.on24.com/eventRegistration/EventLobbyServlet?target=reg20.jsp&eventid=4800134&sessionid=1&key=CB65CFB96D2F8FABD533EBF2187A03C1&groupId=5913544&sourcepage=register" target="_blank" rel="noreferrer noopener">check out our webinar</a> </p> <figure class="wp-block-image size-large"><img decoding="async" width="1024" height="576" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/Size-exclusion-chromatography-2-1024x576.jpg" alt="" class="wp-image-17947" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/Size-exclusion-chromatography-2-1024x576.jpg 1024w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/Size-exclusion-chromatography-2-300x169.jpg 300w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/Size-exclusion-chromatography-2-768x432.jpg 768w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/Size-exclusion-chromatography-2.jpg 1200w" sizes="(max-width: 1024px) 100vw, 1024px" /></figure> <h3 class="wp-block-heading" id="h-separation-of-aav-from-aggregates-by-size"><strong>Separation of AAV from aggregates by size</strong></h3> <p>The success of AAV-based gene therapies depends on <a href="https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bit.27905" target="_blank" rel="noreferrer noopener">the purity and homogeneity of the AAV vector populations</a>, so monitoring AAV aggregation is essential for quality assurance.</p> <p>To identify and distinguish aggregates, you need well-resolved separation power; the stationary-phase particle pores are crucial in this respect.</p> <p>For AAV molecules about 20-25 nm (200-250 脜), the average pore size of your column should ideally range from 50-60 nm (500-600 脜).</p> <h3 class="wp-block-heading" id="h-importance-of-efficient-nbsp-short-sec-columns"><strong>Importance of efficient  short SEC columns</strong></h3> <p>The efficiency of SEC columns plays a vital role in achieving fast and accurate results.</p> <p>Short columns can offer you abundant time-saving advantages; however, they must maintain high efficiency to ensure reliable data. The <a href="https://www.thermofisher.com/order/catalog/product/43903-157831" target="_blank" rel="noreferrer noopener">Thermo Scientific SurePac Bio 550 SEC MDi HPLC column</a> is packed with 3-碌m monodisperse particles, enabling the utilization of short columns without compromising efficiency.</p> <p>This technology allows for the implementation of fast methods without sacrificing the quality and reliability of the analysis.</p> <h3 class="wp-block-heading" id="h-aav-samples-do-not-come-in-abundance"><strong>AAV samples do not come in abundance</strong></h3> <p>AAV samples are often limited in quantity and concentrations. It is essential to use them judiciously and avoid unnecessary large-volume sample volume injections onto your column.</p> <ul class="wp-block-list"> <li>When large volume injection is required, for instance when SEC is coupled to MALS (Multi-Angle Light Scattering) detection, the SurePac Bio 550 SEC MDi column can accommodate large volume injections, without the resolution of the critical pair.</li> </ul> <p>Moreover, your results must be reliable from the first injection without conditioning.</p> <ul class="wp-block-list"> <li>The diol-chemistry of the MDi particles and the inert hardware prevent unwanted secondary interactions that could cause sample loss in the initial injections.</li> </ul> <figure class="wp-block-image size-full"><img decoding="async" width="2155" height="1393" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05.png" alt="" class="wp-image-17952" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05.png 2155w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05-300x194.png 300w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05-1024x662.png 1024w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05-768x496.png 768w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05-1536x993.png 1536w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_05-2048x1324.png 2048w" sizes="(max-width: 2155px) 100vw, 2155px" /></figure> <p><strong><em>Figure 1. Separation of AAV sample with injection volume ranging from 4 to 50 碌L.</em></strong></p> <h3 class="wp-block-heading" id="h-sensitive-detection-with-uhplc-fld-uv-methods"><strong>Sensitive detection with UHPLC-FLD-UV methods</strong></h3> <p><a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-003089-ccs-sec-column-surepac-aav-an003089-na-en.pdf" target="_blank" rel="noreferrer noopener">Sensitive detection</a> is crucial for accurately assessing your AAV samples, since AAV samples are limited in quantity.</p> <ul class="wp-block-list"> <li>Fluorescence detection offers a highly sensitive detection down to the femtogram level while conserving sample volume. By utilizing fluorescence detection, researchers can achieve reliable results while minimizing the amount of AAV sample consumed.</li> </ul> <ul class="wp-block-list"> <li>UV detection, particularly with double wavelength analysis, presents an excellent technique for assessing AAV samples. This approach not only allows for the detection of AAV aggregation at low nano-gram levels, but also provides valuable information on the efficacy based on empty versus full capsid ratios.</li> </ul> <figure class="wp-block-image size-full"><img loading="lazy" decoding="async" width="2106" height="1467" src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c.png" alt="" class="wp-image-17953" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c.png 2106w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c-300x209.png 300w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c-1024x713.png 1024w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c-768x535.png 768w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c-1536x1070.png 1536w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2024/07/fig_02c-2048x1427.png 2048w" sizes="auto, (max-width: 2106px) 100vw, 2106px" /></figure> <p><strong><em>Figure 2</em></strong><em>. Separation of AAV monomer and High Molecular Weight Species (HMWS) using both UV and FLD detection. Black trace represents the UV 280 nm signal, blue trace represents the UV 260 nm signal, and pink trace represents the FLD signal.</em></p> <h3 class="wp-block-heading" id="h-stay-up-to-date-on-analytical-methods-for-bioseparations"><strong>Stay up to date on analytical methods for bioseparations</strong></h3> <p>AAV aggregation analysis by SEC requires the right column to preserve precious samples and achieve sensitive detection.</p> <p>By optimizing sample utilization and implementing efficient short columns, you can streamline fast analyses while maintaining data integrity.</p> <p>Ready to learn more? Watch our webinar: <a href="https://event.on24.com/eventRegistration/EventLobbyServlet?target=reg20.jsp&eventid=4800134&sessionid=1&key=CB65CFB96D2F8FABD533EBF2187A03C1&groupId=5913544&sourcepage=register" target="_blank" rel="noreferrer noopener">Optimizing AAV Analysis with 3 渭m Monodisperse SEC Columns</a></p> <p>Additional Resources:</p> <ul class="wp-block-list"> <li><a href="https://assets.thermofisher.com/TFS-Assets/CMD/manuals/qs-015-ccs-surepac-bio-550-sec-mdi-qs015-na-en.pdf" target="_blank" rel="noreferrer noopener">SurePac Bio 550 SEC MDi quick start guide</a></li> <li><a href="https://assets.thermofisher.com/TFS-Assets/CMD/manuals/man-015-ccs-surepac-bio-550-sec-mdi-man015-na-en.pdf" target="_blank" rel="noreferrer noopener">SurePac Bio 550 SEC MDi columns product manual</a></li> </ul> <p>Visit our <a href="https://www.linkedin.com/showcase/chromatography-&-mass-spectrometry/posts/?feedView=all" target="_blank" rel="noreferrer noopener">LinkedIn page</a>: #AAV #UHPLC</p> </div></article><section class="author-box"><h4 class="author-box-title">Mauro De Pra</h4><div class="author-box-content" itemprop="description">Mauro De Pra is a HPLC Product Marketing Manager at Thermo Fisher Scientific. With a tenure of over a decade at the company since joining in 2010, he has focused on HPLC product development, application development and support. Prior to his role at Thermo Fisher Scientific, Mauro served as an Analytical Development Scientist at Merck Sharp & Dohme. Mauro's academic background includes a Ph.D. in analytical chemistry from the University of Amsterdam in The Netherlands </div></section><div class="adjacent-entry-pagination pagination"><div class="pagination-previous alignleft"><i class="fa fa-icon fa-chevron-left"></i> <a href="https://www.thermofisher.com/blog/analyteguru/the-vital-role-of-ic-in-ensuring-water-quality/" rel="prev">The Vital Role of IC in Ensuring Water Quality</a></div><div class="pagination-next alignright"><a href="https://www.thermofisher.com/blog/analyteguru/consider-semi-preparative-columns-to-improve-your-oligonucleotide-analysis/" rel="next">Consider Semi-preparative Columns to Improve Your Oligonucleotide Analysis</a> <i class="fa fa-icon fa-chevron-right"></i></div></div></main></div></div><footer class="site-footer"><div class="wrap"><div class="creds"><p class="footer-links"><a href="https://corporate.thermofisher.com/en/footer/privacy-statement.html" target="_blank" rel="noopener noreferrer">Privacy Statement</a><a href="https://corporate.thermofisher.com/en/footer/terms-and-conditions.html" target="_blank" rel="noopener noreferrer">Terms & Conditions</a><a href="/blog/locations/">Locations</a><a href="/blog/sitemap/">Sitemap</a></p><p>© 2025 <a href="https://www.thermofisher.com">Thermo Fisher Scientific</a>. 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