News from LabRulezLCMS Library - Week 22, 2025

<p><strong>When I think about what it takes to keep a high-performing MS laboratory running—especially those dealing with tight timelines, strict regulations, and ever-expanding datasets—I’m reminded that the software we rely on isn’t just a tool. It’s the backbone of our workflows, our decisions, and ultimately, our results.</strong></p><p>Whether you’re in pharma, environmental testing, or food safety, managing the complexity of targeted mass spectrometry workflows while maintaining accuracy, compliance, and efficiency requires a lot of work.</p><p>That’s where a purpose-built solution makes a difference. I’ve worked closely with labs that are navigating these challenges—and from the many conversations and experiences, I put together five main capabilities you should look for in your software when it comes to targeted MS quantitation:</p><h3><strong>1. Simplified data and workflow management</strong></h3><p>Too many labs are still relying on disconnected systems or folder-based setups that can lead to inconsistent methods and reports. When utilizing modern software solutions, everything—methods, templates, data, reports—can be centrally managed, reducing errors and training time while ensuring everyone is working from the same validated workflows.</p><p>It’s a big plus if the software also supports multi-consensus reporting, making it easy to compare results across sequences and analyses for greater confidence in the data. By packaging complete workflows and setting predefined views, labs can streamline routine operations and ensure method repeatability, so anyone can run the analysis reliably and consistently.</p><h3><strong>2. Controlled access in and out of the lab</strong></h3><p>Many labs are still tied to legacy setups that make remote work challenging. Researchers often rely on standalone workstations that store data locally, making it hard to access critical results when off-site. Transferring data via USB drives or external hard disks not only adds inefficiency, but it can also raise serious security concerns. These outdated practices slow down collaboration and increase the risk of data loss or breaches.</p><p>Hybrid work is here to stay. With the ability to control and access instruments and data, users can securely set up runs, review results, and manage workflows from anywhere. You’re no longer tied to a specific bench, workstation, or even building. Productivity doesn’t have to stop when you step out of the lab.</p><p><i>“Managing all our instruments and work with data remotely via a centralized server means we don’t need to be in the laboratory to access our data.” – Dr. Kayleigh Arthur, Owlstone Medical Ltd.</i></p><h3><strong>3. It’s built for MS workflows</strong></h3><p>Whether you’re running triple quads, high-res accurate mass, or both—life’s much easier when working with software solutions that give you native instrument control and deep integration for targeted workflows. That includes support for MSⁿ, intact mass deconvolution, spectral libraries, and high-throughput quantitation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_9e5e9dc842.jpg" alt="Thermo Fisher Scientific: 5 Things to Consider When Selecting Software for Targeted MS Quantitation." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_9e5e9dc842.jpg 245w," sizes="100vw" width="1536" height="411"></p><p>Just as important is what happens after data acquisition. Integrated, built-in reporting capabilities—paired with customizable reporting options—should make it easy to flag results and automate the necessary calculations, all within the software. You need a powerful combination that can streamline review and ensures consistency across analyses.</p><h3><strong>4. Scalability</strong></h3><p>From small bioanalytical teams to global enterprises, can it be scaled up or down without the need for reinstallation or manual data transfer? It should be built to handle complex, multi-site deployments with ease.</p><p>Add to that network resilience, automated data protection, and centralized updates—and you’ll get a system designed to grow with your organization.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_2_8ec325678b.jpg" alt="Thermo Fisher Scientific: 5 Things to Consider When Selecting Software for Targeted MS Quantitation." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_2_8ec325678b.jpg 245w," sizes="100vw" width="1200" height="660"></p><article><div><h3><strong>5. Stress-free compliance</strong></h3><p>Let’s face it, if you have to do it, compliance is non-negotiable, but it can be time-consuming. The system should include built-in features that support GxP, 21 CFR Part 11, and audit readiness—like audit trails, version control, and detailed user management.</p><p>The ability to query and interrogate data within the system would ensure that data integrity requirements are met, delivering full traceability, and verification of data throughout its lifecycle. Your QA team will be very pleased!</p><h3><strong>Conclusion</strong></h3><p>Managing targeted MS quantitation shouldn’t feel like assembling a puzzle with missing pieces. A modern software should bring together everything you need—scalable infrastructure, powerful MS data tools, and built-in compliance features—into one cohesive solution. It must support your science, your team, and your business goals.</p><p>If you’re ready to rethink how your lab handles MS data, this is the right time to explore what’s possible.</p><blockquote><p>Join our <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5232"><strong>upcoming webinar</strong></a> to learn about networked software for MS labs.</p><p><strong>Visit us on&nbsp;</strong><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/"><strong>LinkedIn:</strong></a>&nbsp;#targetedquantitation #compliance #massspectrometry #control</p></blockquote></div></article><section><blockquote><h4><strong>Laercio Fernandes</strong></h4><p>Laércio Fernandes is a Product Marketing Manager for software at Thermo Fisher Scientific. With a background rooted in analytical chemistry, Laércio has over 20 years of experience in the analytical field with a focus on chromatography instrumentation, enterprise software and compliance. His background in sales and support has enabled him to have a good overview and understanding of different processes and challenges that laboratories may face, which has allowed him to better support our customers in order to improve their workflows and productivity.</p></blockquote></section>

5 Things to Consider When Selecting Software for Targeted MS Quantitation

Characterisation of small RNA-based therapeutics and their process impurities by fast and sensitive liquid chromatography high resolution mass spectrometry

<p><strong>In the world of biotherapeutics, bigger often means better — at least when it comes to circulation time in the bloodstream. One of the most widely used strategies to extend the half-life of protein drugs is PEGylation: attaching polyethylene glycol (PEG) chains to a therapeutic protein. The added polymer shields the protein, reduces immune clearance, and improves pharmacokinetics.</strong></p><h4><strong>PEGylation creates a major analytical challenge</strong></h4><p>PEG is <strong>large and polydisperse</strong> (a distribution of chain lengths), and when attached to already complex, glycosylated proteins, it creates a molecular puzzle. How many PEGs are attached? Where are they attached? Are there impurities left behind?</p><p>In this study, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Charge Detection Mass Spectrometry and a Glu-C/Lys-C Digestion-Based Data-Dependent Approach Suggest Mono-PEGylation of a Heterogenous Therapeutic Protein</strong></a>, researchers developed a powerful mass spectrometry workflow that answers these questions — using Orbitrap-based charge detection mass spectrometry and a clever bottom-up strategy. The result? A confident determination that the therapeutic protein carries <strong>a single ~30 kDa PEG</strong>, along with precise mapping of its attachment sites.</p><p>Let’s break down how they did it.</p><h3><strong>The analytical problem: pegylation meets glycosylation</strong></h3><p><strong>The therapeutic protein studied here is already highly heterogeneous:</strong></p><ul><li><strong>165 </strong>amino acids</li><li><strong>3 </strong>N-glycosylation sites</li><li><strong>1 </strong>O-glycosylation site</li></ul><p>Add a <strong>~30 kDa polydisperse PEG </strong>on top of that, and conventional mass spectrometry quickly runs into trouble:</p><ul><li>PEG ionizes very efficiently, masking protein charge states</li><li>Charge envelopes overlap</li><li>Intact mass deconvolution becomes difficult</li><li>PEGylated peptides fragment poorly</li></ul><p>Traditional approaches like post-column triethylamine (TEA) addition or MALDI have <strong>limitations</strong>, especially when glycosylation adds another layer of complexity.</p><p>This is where <strong>Orbitrap-based Charge Detection Mass Spectrometry (CD-MS)</strong> changes the game.</p><h3><strong>Step 1: Measuring the intact mass with charge detection MS</strong></h3><p>The <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>team</strong></a><strong> </strong>used a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/560"><strong>Thermo Scientific Q Exactive UHMR</strong></a> instrument operating in <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/direct-mass-technology.html"><strong>Direct Mass Technology mode</strong></a>.</p><p><strong>Unlike conventional ensemble MS, CD-MS measures:</strong></p><ul><li>The m/z of each individual ion</li><li>The charge of each ion directly</li></ul><p>This enables direct mass determination of highly heterogeneous species.</p><h5><strong>What did they see?</strong></h5><p><strong>From the data:</strong></p><ul><li><strong>Non-PEGylated protein (TP):</strong> ~28.95 kDa</li><li><strong>PEGylated protein (PEG-TP):</strong> ~58.42 kDa</li></ul><p>That mass shift strongly supports <strong>mono-PEGylation</strong> — one PEG per protein molecule.</p><p>Even more impressively, <strong>CD-MS revealed:</strong></p><ul><li>A minor dimer impurity (~3.7%)</li><li>A small free PEGylation reagent (~30.4 kDa), likely residual reagent</li></ul><p>The mirror plot (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Figure 2 on page 4</strong></a>) visually highlights this mass shift, clearly separating the TP and PEG-TP populations.</p><h4><strong>Why CD-MS was critical</strong></h4><p>Because PEG is polydisperse, it broadens the mass distribution. In conventional ensemble MS, overlapping charge states prevent proper deconvolution. CD-MS bypasses this by assigning charge per ion, enabling:</p><ul><li>Accurate intact mass measurement</li><li>Impurity detection</li><li>Quantification</li><li>High reproducibility across runs</li></ul><p>For complex PEGylated biologics, this represents a<strong> major analytical advance</strong>.</p><h3><strong>Step 2: Mapping the pegylation sites (bottom-up strategy)</strong></h3><p>Knowing there’s one PEG is not enough — regulators and developers need to know <strong>where</strong> it’s attached.</p><p><strong>The team designed a multi-enzyme digestion workflow:</strong></p><ul><li>Lys-C digestion</li><li>Glu-C digestion</li><li>Lys-C/Glu-C combination</li><li>PNGase F treatment to remove N-glycans</li></ul><p>Each digestion generated peptides of different lengths, providing complementary information.</p><p>But there was still a problem.</p><p>PEGylated peptides are difficult to fragment because the large PEG dominates ionization and masks peptide signals.</p><h3><strong>The clever trick: high source CID</strong></h3><p>They applied <strong>high in-source collision-induced dissociation (~90 eV)</strong> to partially strip PEG chains before MS/MS.</p><p>This generated truncated PEG forms (PEG1–PEG50 variants), allowing:</p><ul><li>Precursor isolation</li><li>Efficient HCD fragmentation</li><li>Identification of diagnostic fragment ions</li></ul><p><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Figure 4 (page 7)</strong></a><strong> </strong>dramatically shows how high source CID transforms a messy PEG-dominated spectrum into clean multiply charged peptide precursors ready for sequencing.</p><h3><strong>The result: three pegylation sites identified</strong></h3><p>Using DDA and software-assisted analysis (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1837"><strong>Biopharma Finder</strong></a>), the study confidently identified PEGylation at:</p><ul><li><strong>K52 (major site)</strong></li><li><strong>K45</strong></li><li><strong>N-terminal A1</strong></li></ul><p><strong>K52 — The dominant site</strong><br>K52 was consistently observed across all digestion strategies. Fragment maps showed strong diagnostic ions carrying truncated PEGs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Figures 5–7</strong></a>).<br>Multiple truncations (PEG22–PEG29 and beyond) were observed, confirming robust assignment.</p><p><strong>K45 — A secondary site</strong><br>K45 was confirmed in the combined Lys-C/Glu-C digestion (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Figure 8</strong></a>), where shorter peptides improved fragmentation and site localization.</p><p><strong>N-Terminal A1 — also modified</strong><br>Unexpectedly, PEGylation was also identified at the N-terminus (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Figure 9</strong></a>), with highly accurate fragment ion mass measurements supporting this assignment.</p><p><strong>The evidence suggests that:</strong></p><ul><li>K52 and A1 are the most frequently occupied sites</li><li>K45 is less populated</li></ul><h3><strong>Why this matters for biopharma</strong></h3><p><strong>PEGylated therapeutics must demonstrate:</strong></p><ul><li>Batch-to-batch consistency</li><li>Controlled PEGylation stoichiometry</li><li>Clear impurity profiles</li><li>Verified modification sites</li></ul><p><strong>This workflow delivers:</strong></p><ul><li><strong>Intact mass confirmation under native condition</strong></li><li><strong>Quantification of impurities</strong></li><li><strong>Confident site localization</strong></li><li><strong>High reproducibility</strong></li><li><strong>Software-assisted interpretation</strong></li></ul><p>Importantly, it requires minimal sample — a key advantage in early development.</p><h3><strong>The bigger picture: a blueprint for complex biologics</strong></h3><p><strong>The combination of:</strong></p><ul><li>Orbitrap-based Charge Detection MS</li><li>Native intact mass analysis</li><li>Multi-enzyme digestion</li><li>High source CID optimization</li><li>Advanced data processing</li></ul><p>creates a seamless, comprehensive characterization strategy.</p><p>Since pharma companies have been adding PEGylated modalities to their pipelines, the importance of this study will continue to shine. Furthermore, PEGylation and other polymer conjugations continue to evolve, and the analytical workflows must keep pace. This study demonstrates how modern mass spectrometry — particularly single-ion charge detection — can tackle extreme molecular heterogeneity with confidence.</p><p>For PEGylated therapeutics, the mass spectrometer doesn’t just weigh molecules. It tells the full molecular story.</p><blockquote><h4><strong>Reference:</strong></h4><p><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00788?goto=supporting-info"><strong>Charge Detection Mass Spectrometry and a Glu-C/Lys-C Digestion-Based Data-Dependent Approach Suggest Mono-PEGylation of a Heterogenous Therapeutic Protein</strong></a></p><p>Zoltan Szabo, Shero Lao, Katie S. Peterson, Kristina Srzentič, Kyle P. Bowen, Amanda E. Lee, Michael W. Senko, Dietmar Reusch, Steffen Lippold, and Markus Haberger</p><p><i>Journal of Proteome Research</i> <strong>Article ASAP</strong></p><p>DOI: 10.1021/acs.jproteome.5c00788</p></blockquote><p><strong>Visit us on&nbsp;</strong><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/"><strong><u>LinkedIn:</u></strong></a>&nbsp;#PEGylation #Pharmacuetical #teamMassSpec #therapeutics</p>

Cracking the Code of PEGylated Proteins: How Advanced Mass Spectrometry Reveals What’s Really Attached

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 3rd August 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Shimadzu / ASMS, Thermo Fisher Scientific / HPLC Symposium </strong>and<strong> Waters Corporation / ASMS </strong>and application note by<strong> Metrohm!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Ultra-Trace Quantification of N-Nitroso-desmethyl-sumatriptan with Interference Resolution Using LC–TQ</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/1290_infinity_iii_uhplc_6495d_lc_tq_desmethyl_sumatriptan_asms_2026_mp_667_agilent_ebb0888227.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Sumatriptan succinate, an antimigraine drug contains nitrosylating functional groups and is susceptible to forming its own nitrosamine drug substance-related impurity (NDSRI), specifically N-nitroso-desmethylsumatriptan. As a Category 1 genotoxic impurity, its specification limit is established at 0.0428 ppm with respect to the API. In this study, samples and standards were prepared in methanol:water (1:1, v/v). The LC/TQ method was developed with a target limit of detection (LOD) at least 20-fold lower than the specification limit to ensure adequate sensitivity. Analytical data were collected in replicate for a 10 mg/mL API sample, as well as LOQ- and specification-level spiked samples, to evaluate recovery, relative standard deviation (RSD), linearity, and repeatability.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+III+UHPLC"><strong>1290 Infinity III UHPLC</strong></a></li><li><strong>MS:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D"><strong>6495D LC/TQ</strong></a></li><li><strong>Column:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Poroshell+120+EC+C18"><strong>Poroshell 120 EC C18</strong></a>, 3x150mmx2.7µm, 40 Deg C</li></ul><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>A sensitive and robust method has been developed for N-nitroso-desmethyl Sumatriptan in Sumatriptan.&nbsp;</li><li>Method LOQ is 10% lower than specification level, RSD of &lt;5%, and linearity across 0.00214 to 0.1 ppm.&nbsp;</li><li>The recovery of NDSRI in Sumatriptan at LOQ level and specification level are 95%.</li></ul><h3><strong>2. Metrohm: Moisture analysis of rebaudioside M by Karl Fischer titration</strong></h3><p><strong>Simplified rebaudioside M moisture determination via volumetric Karl Fischer titration paired with an oven system</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_k_075_download_59f185c238.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Natural sweeteners like stevia and its derivatives are an increasingly important part of the global sweetener market as more consumers substitute sugar with healthier alternatives. Stevia’s derivatives are significantly sweeter than table sugar. These compounds are estimated to be 50 to 300 times sweeter in comparison. This sweet taste coupled with the fact that the human body cannot metabolize steviol glycosides (i.e., they contribute zero calories to one’s diet), makes them a very attractive sugar replacement.&nbsp;</p><p>The moisture content of stevia materials is a crucial factor when discussing their shelf life and stability. Measuring the moisture content of a stevia derivative, rebaudioside M, using an oven system coupled with volumetric Karl Fischer (KF) titration provided a repeatable approach for quantifying the moisture content of this material.</p><h4><strong>Experimental</strong></h4><p>The analysis was carried out with a Double Pt ring electrode for volumetry (6.00346.100). Testing was executed on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=220&amp;search=874+Oven+Sample+Processor"><strong>874 Oven Sample Processor</strong></a> (2.874.0120) coupled with an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=220&amp;search=852+Titrando"><strong>852 Titrando</strong></a> (2.852.0060). Approximately 0.2 g of sample was weighed into an 874 Oven Sample Processor 6 mL vial and placed onto the rack. The oven was set to a temperature of 100 °C, and the evaporated moisture was carried to the volumetric titration vessel by nitrogen gas flow at 70 mL/min. The moisture from the sample was titrated with Hydranal Comp 5 reagent.</p><h4><strong>Conclusion</strong></h4><p>Natural low-calorie sweeteners like stevia derivatives are growing in popularity as people choose healthier alternatives to sugar. Working to develop standardized, specific methodology can help support sweetener industry development, effective quality assessment, and the evaluation of rebaudioside M and other steviol glycosides’ stability.&nbsp;</p><p>This Application Note provides a foundation for the analysis of stevia powder by Karl Fischer titration. An oven system paired with volumetric Karl Fischer analysis provides an accurate, repeatable method to evaluate stevia powder and its derivatives. This method’s compatibility with the 874 Oven Sample Processor allows increased sample throughput through automation, providing operators even more time to complete other important tasks in parallel.</p><h3><strong>3. Shimadzu / ASMS: Quantitative analysis of 6 beta blockers on an LCMS-8045RX</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/WP_131_Quantitative_analysis_of_6_beta_blockers_on_an_LCMS_8045_RX_f596f609f4.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Beta-adrenergic blocking agents, commonly known as beta blockers, competitively inhibit the binding of catecholamines such as epinephrine and norepinephrine to β-adrenergic receptors, thereby reducing heart rate and blood pressure. These drugs are widely prescribed for the treatment of hypertension, angina and heart failure<sup>1</sup> . However, beta blockers may also be misused by athletes resulting in their inclusion on the World Anti-Doping Agency (WADA) prohibited list<sup>2</sup> . Owing to both their therapeutic importance and potential misuse, sensitive and selective analytical methods are required for reliable detection and quantification. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers high sensitivity and specificity for such analyses.</p><h4><strong>Method</strong></h4><p>Nadolol, metoprolol, propranolol, atenolol &amp; carvedilol were purchased from Sigma Aldrich and Thermo Fisher Scientific. Standards were prepared in 1 mg/mL in LCMS grade methanol. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+40"><strong>Nexera 40-series HPLC system</strong></a> connected to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8045RX"><strong>LCMS-8045RX</strong></a> was used for data acquisition. The mixture of standards was further diluted in LCMS grade methanol (Sigma Aldrich) and Sigmatrix urine diluent to create the standards for the calibration curves. A Shim-pack Velox Biphenyl column was used for the chromatographic separation. All analytes were ionized with electrospray ionization (ESI) in positive ion mode.</p><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>Superior sensitivity in complex matrices: Limit of Detection (at accuracy ±20% and S/N &gt; 3) of 0.010 ng/mL for all the analytes evaluated in this study.</li><li>Excellent linearity and accuracy: Linear responses (with R2 &gt; 0.99) observed across the evaluated concentration range, and consistent accuracy within ±20% for all 17 calibrants included in this study.&nbsp;</li><li>Optimized throughput: Automated interface and MRM optimization via LabSolutions Connect MRM ensured maximum signal-to-noise determination for this study, while the high-organic flush at the end of each run, maintained column integrity</li></ul><h3><strong>4. Thermo Fisher Scientific / HPLC Symposium: A switchable 2D-LC platform in PAT: routine UV monitoring or MS-based characterization of mAb PQAs directly from the bioreactor</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_004764_hplc_vanquish_2dlc_mabs_analysis_bioreactor_samples_po004764_na_en_7a99c1b683.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Monitoring quality attributes throughout the upstream processing of mAbs is critical due to potential effects of biological variability on product safety and efficacy. Thus, there is a growing need for streamlined analytical workflows that provide timely and reliable insights to enable fast and informed process decisions.</p><h4><strong>Materials and methods</strong></h4><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Thermo Scientific Vanquish Flex Simple</strong></a> Switch 2D-LC system for loop heart-cutting consisting of Quaternary Pump F (VF-P20-A), Binary Pump F (VF-P10-A-01), Dual Split Sampler FT (VF-A40-A-02), 2x Column Compartment H (VH-C10-A-03), and Diode Array Detector FG (VF-D11-A-01)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1490"><strong>Thermo Scientific Orbitrap Exploris MX Mass Detector</strong></a> (BRE725536) with BioPharma option (BRE725539)&nbsp;</li><li>Thermo Scientific MAbPac Protein A column, 12 µm, 4 x 35 mm (PN 082539)&nbsp;</li><li>Thermo Scientific ProPac 3R SCX, 2 x 50 mm, 3 µm (PN 43103-052068)&nbsp;</li><li>Thermo Scientific MAbPac SEC-1, 5 µm, 4 x 300 mm (PN 074696)</li></ul><h4><strong>Data analysis&nbsp;</strong></h4><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon 7.3.2 Chromatography Data System (CDS) </strong></a>and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1837"><strong>Thermo Scientific BioPharma Finder 5.2 software</strong></a></p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Switchable 2D-LC-UV/MS offers a streamlined approach for at-line monitoring of mAb titer, charge and size variants directly from the bioreactor in &lt;1h.&nbsp;</li><li>UV monitoring provides quick and simple trendlines, while HRAM-MS allows deep characterization at intact level.&nbsp;</li><li>Near real-time information supports in-time process decisions to protect yield and product quality.</li></ul><h3><strong>5. Waters Corporation / ASMS: SOFTWARE TOOLS FOR AUTOMATED LC/MS ANALYSIS OF CRITICAL QUALITY ATTRIBUTES OF mRNA MOLECULES</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009414_52de0bb71a.pdf"><strong>Full PDF for download</strong></a></li></ul><div><div><section><div><div><div><div><div><div><p>This poster presents an automated LC-MS workflow for evaluating three critical quality attributes (CQAs) of mRNA therapeutics: <strong>5′-capping efficiency, Poly(A) tail length and heterogeneity, and mRNA sequence integrity</strong>. A custom firefly luciferase (Fluc) mRNA construct containing a Cap-1 structure, 1,919 nucleotides, and a Poly(A) tail was used as the model sample. Importantly, all three CQAs were assessed using a <strong>single LC-MS assay</strong>, supported by specialized waters_connect software tools designed to streamline nucleic acid characterization from sequence entry through final data processing.</p><p>The analytical platform consisted of a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>Waters ACQUITY Premier Binary UPLC System</strong></a><strong> with TUV detector coupled to a </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Xevo MRT Q-Tof Mass Spectrometer</strong></a>, which uses multi-reflectron time-of-flight technology. Separations were performed on an ACQUITY Premier OST 1.7 µm, 300 Å, 2.1 × 150 mm column, while data-independent acquisition was carried out in MSE mode. Data processing used the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544"><strong>waters_connect</strong></a><strong> Informatics Platform together with SYNTHETIC Library 2.0, MAP Sequence 2.0, and INTACT Mass 1.9</strong> applications. For sequence mapping, the mRNA was independently digested using four endonucleases—RNase T1, RapiZyme MC1, RapiZyme Cusativin, and human RNase4—providing complementary cleavage patterns.</p><p>The workflow produced detailed information on both capping and Poly(A) tail characteristics. The Fluc mRNA showed <strong>100% capping efficiency</strong>, with 62.7% carrying an m7GpppAm Cap-1 structure and 37.3% an m7GpppGm Cap-1 structure; no uncapped digestion product or Cap-related impurities were detected. Poly(A) tail analysis identified ten major species ranging from <strong>98-mer to 107-mer</strong>, resulting in an average Poly(A) tail length of <strong>102.3 nucleotides</strong>. The Xevo MRT Q-Tof provided high-resolution measurements—approximately 100,000 resolution in the examples shown—and very good mass accuracy for the detected Poly(A) species.</p><p>For sequence integrity, combining the four complementary enzymatic digestions achieved <strong>93.7% unambiguous mRNA sequence coverage</strong>, demonstrating the value of a multi-enzyme approach for comprehensive sequence confirmation. MAP Sequence 2.0 also automatically distinguished structural isomers using elevated-energy fragment-ion information, helping resolve isobaric and isomeric digestion products with less manual interpretation. Overall, the poster demonstrates that high-resolution MSE acquisition on the <strong>Xevo MRT Q-Tof combined with automated waters_connect informatics</strong> can provide an integrated and efficient approach for comprehensive characterization of key mRNA quality attributes.</p></div></div></div></div></div></div></section></div></div>

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