Scientists at IOCB Prague are uncovering new details of gene transcription. They have identified a previously unknown molecular mechanism by which the transcription of genetic information from deoxyribonucleic acid (DNA) into ribonucleic acid (RNA) can be initiated. The researchers focused on a specific class of molecules known as alarmones, which are found in cells across a wide range of organisms and whose levels often increase under conditions of cellular stress. The results were published in the prestigious scientific journal Nature Chemical Biology.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_bc2bfffa6c.jpg" alt="IOCB Prague/Tomáš Belloň: Research from IOCB Prague reveals a previously unknown mechanism of genetic transcription: Dr. Hana Cahová, head of the Chemical Biology of Nucleic Acids research group." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_bc2bfffa6c.jpg 234w," sizes="100vw" width="2000" height="1335"><div>Molecules of ribonucleic acid can carry a variety of chemical modifications at one end, known as caps. In eukaryotic organisms, including human cells, the best-known cap plays an important role in RNA stability and in regulating its subsequent fate. In recent years, however, it has become clear that alternative, non-canonical RNA caps also exist, although their formation and the mechanisms by which they are attached to RNA remain only partially understood. These include alarmone caps, formed by dinucleoside polyphosphate molecules, that protect cellular RNA at moments when the cell is under threat.In their study, <a target="_blank" rel="noopener noreferrer" href="https://cahova.group.uochb.cz/en">Dr. Hana Cahová</a> and colleagues examined bacterial RNA polymerase and investigated how this enzyme can initiate transcription using dinucleoside polyphosphates (NpNs) instead of the standard RNA building blocks. For the first time, the scientists described, at the atomic level, how RNA bearing an alarmone cap can be generated directly at the initiation of gene transcription. They also observed that NpNs bind through a different type of base pairing than is typically observed.The work was significantly shaped by Valentina Serianni from Hana Cahová’s team, who demonstrated the ability of dinucleoside polyphosphates to initiate gene transcription, and by Jana Škerlová, who focused on the structural analysis of RNA polymerase. Using cryogenic electron microscopy data, she showed how dinucleoside polyphosphate molecules bind within the active site of RNA polymerase – that is, the core of the enzyme where genetic information is transcribed. Together, their findings help to elucidate the processes that accompany the transcription of genetic information from DNA to RNA.Hana Cahová adds: “We’re describing something that truly occurs in cells and that we’re now able to observe directly at the level of individual molecules. This allows us to answer fundamental questions about cellular processes, such as how cells adapt to stress. RNA plays a central role in this, as it carries the cascade of information underlying any cellular response – for example, to threatening conditions caused by nutrient deprivation or temperature shock.”<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_2_0fe88ff9e9.jpg" alt="IOCB Prague/Tomáš Belloň: Research from IOCB Prague reveals a previously unknown mechanism of genetic transcription: Dr. Tomáš Kouba, head of the Cryogenic Electron Microscopy core facility." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_2_0fe88ff9e9.jpg 234w," sizes="100vw" width="2000" height="1335"><div>Cryogenic electron microscopy (cryo-EM) was central to the project. This part of the work was led by Dr. Tomáš Kouba, one of the authors of the study. “Cryogenic electron microscopy allows us to freeze biological molecules in a state very close to their natural form and then determine their three-dimensional structure. This makes it possible to look directly into the active centers of enzymes and observe their function down to the atomic level,” he explains.Last year, IOCB Prague opened a new <a target="_blank" rel="noopener noreferrer" href="https://cryoem.group.uochb.cz/en">cryo-EM center</a> that is unique within the Czech research environment. State-of-the-art cryogenic electron microscopes are housed in a specially designed building and enable the study of biological processes with exceptional structural precision.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_3_4d881e7a58.jpg" alt="IOCB Prague: Research from IOCB Prague reveals a previously unknown mechanism of genetic transcription: This work shows how NpnNs initiate bacterial transcription. Cryo-EM reveals how nucleobases pair with the template: one in canonical and one in reverse Watson–Crick mode, providing a structural basis for their function as non-canonical RNA caps." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_UOCHB_Vyzkum_z_UOCHB_odhaluje_dosud_nepoznany_zpusob_prepisu_geneticke_informace_3_4d881e7a58.jpg 245w," sizes="100vw" width="2000" height="1077"><blockquote><h4>The Original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.nature.com/articles/s41589-025-02134-5">Molecular insight into 5′ RNA capping with NpnNs by bacterial RNA polymerase</a></h4>Valentina M. Serianni, Jana Škerlová, Anna Knopp Dubánková, Anton Škríba, Hana Šváchová, Tereza Vučková, Anatolij Filimoněnko, Milan Fábry, Pavlína Řezáčová, Tomáš Kouba &amp; Hana Cahova&nbsp;Nat. Chem. Biol. 21 (2026)<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1038/s41589-025-02134-5">https://doi.org/10.1038/s41589-025-02134-5</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote><h4>Abstract</h4>RNA capped with dinucleoside polyphosphates has been discovered in bacteria and eukaryotes only recently. The likely mechanism of this specific capping involves direct incorporation of dinucleoside polyphosphates by RNA polymerase as noncanonical initiating nucleotides. However, how these compounds bind into the active site of RNA polymerase during transcription initiation is unknown. Here, we explored transcription initiation in vitro, using a series of DNA templates in combination with dinucleoside polyphosphates and model RNA polymerase from Thermus thermophilus. We observed that the transcription start site can vary on the basis of the compatibility of the specific template and dinucleoside polyphosphate. Cryo-electron microscopy structures of transcription initiation complexes with dinucleoside polyphosphates revealed that both nucleobase moieties can pair with the DNA template. The first encoded nucleotide pairs in a canonical Watson–Crick manner, whereas the second nucleobase pairs noncanonically in a reverse Watson–Crick manner. Our work provides a structural explanation of how dinucleoside polyphosphates initiate RNA transcription.</div></div>

Research from IOCB Prague reveals a previously unknown mechanism of genetic transcription

Food adulteration is a phenomenon with a long history that has gained increasing importance in recent years due to the globalization of food supply chains and strong economic pressures. Whether it involves diluting olive oil with cheaper oils, adding sugar to honey, or the hidden use of substitutes in protein supplements, the result is a loss of consumer trust and, in some cases, a potential risk to health. Analytical chemistry plays a key role in this area by providing methods and technologies capable of identifying and quantifying target compounds as well as unwanted impurities in a wide range of food matrices.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Watrex_Praha_Falsovani_potravin_a_jak_ho_odhali_analyticka_chemie_6912db5492.jpg" alt="Watrex: Food Adulteration and How It Is Revealed by Analytical Chemistry" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Watrex_Praha_Falsovani_potravin_a_jak_ho_odhali_analyticka_chemie_6912db5492.jpg 245w," sizes="100vw" width="1600" height="900"><h4>The Role of Chromatography in Forensic Food Analysis</h4>Chromatography, particularly liquid chromatography (HPLC), is one of the most effective analytical tools for separating complex mixtures of molecules. It enables:<ul><li>qualitative identification of individual components in a sample,</li><li>quantitative determination of their concentrations,</li><li>detection of impurities and unexpected components that may indicate adulteration.</li></ul>In practice, HPLC is often combined with various types of detectors (UV/VIS, DAD, refractive index detectors) or with other techniques (e.g., mass spectrometry) to increase selectivity and sensitivity.<h4>Isocratic vs. Gradient Elution</h4>In the analysis of food samples, chromatographic methods are selected according to the analytical goal:<ul><li>Isocratic HPLC uses a constant mobile phase composition throughout the analysis. It is simple, robust, and ideal for routine determinations of well-defined target analytes (e.g., sugars or vitamins).</li><li>Gradient HPLC changes the mobile phase composition during separation, enabling better resolution of complex mixtures with a wide range of polarities (e.g., peptides or phenolic compounds).</li></ul>Gradient methods are particularly useful when analytes have very different properties and high separation efficiency is required.<h4>Watrex Praha and the Contribution of Modern Systems to Food Analysis</h4><a target="_blank" rel="noopener noreferrer" href="https://watrex.com/?utm_source=labrulez&amp;utm_medium=propagace&amp;utm_campaign=labrulez_clanky_HPLCsystemy">Watrex Praha, s.r.o.</a> is one of the leading Czech manufacturers and distributors of liquid chromatography systems and instruments, with a long tradition dating back to 1990. The company has successfully transformed from a distributor into a manufacturer of its own HPLC and related technologies, focusing on modular, robust systems suitable even for demanding analytical tasks.Their <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?manufacturerItems=93&amp;search=streamline">Streamline™ product line</a> includes several HPLC system concepts suitable for food analysis:<ul><li>Isocratic HPLC system – a simple yet precise configuration with a single pump, column thermostat, and UV/VIS detector; suitable for routine determination of known analytes (e.g., basic carbohydrates, food additives).</li><li>Binary gradient HPLC system – equipped with two pumps enabling mobile phase gradient formation, which enhances separation performance for complex mixtures (e.g., phenolic profiles of wine, components of aromatic extracts).</li><li>GPC/SEC system (Gel Permeation / Size Exclusion Chromatography) – a specialized solution for the determination of macromolecules and polymers, also relevant for the analysis of high-molecular-weight components in foods (e.g., polysaccharides).</li></ul><blockquote>👉<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Watrex_catalog_2024_ba97395f92.pdf">Product catalogue HERE</a></blockquote>Thanks to the modular nature of these systems, analysts can easily adapt the configuration to a specific analytical problem, from simple routine analyses to complex characterization of food samples.<h4>Practical Examples of Adulteration Revealed by HPLC</h4><ul><li>Detection of olive oil dilution: HPLC can separate and quantify characteristic triacylglycerols and fatty acids; unusual patterns indicate the presence of cheaper oils.</li><li>Estimation of sugar addition in honey: Isocratic HPLC with a refractive index detector can determine sugar profiles (glucose, fructose, sucrose), revealing dilution or the addition of sugar syrups.</li><li>Identification of aromatic adulterants in wine: Gradient HPLC with a PDA detector can reveal phenolic and aromatic profiles that are altered by the addition of synthetic compounds.</li></ul><h4>Conclusion</h4>Food adulteration remains a current issue that requires reliable and sensitive analytical methods. Liquid chromatography, and in particular modern HPLC systems, represent one of the most powerful tools for detecting unwanted additives, impurities, and unexpected components in complex food samples. Manufacturers such as <a target="_blank" rel="noopener noreferrer" href="https://watrex.com/?utm_source=labrulez&amp;utm_medium=propagace&amp;utm_campaign=labrulez_clanky_HPLCsystemy">Watrex Praha</a> provide flexible chromatographic platforms covering a wide range of analytical tasks from simple isocratic to advanced gradient and GPC/SEC applications thereby supporting analysts in ensuring food quality and consumer protection.<blockquote>Did you find this article interesting? Follow <a target="_blank" rel="noopener noreferrer" href="https://watrex.com/?utm_source=labrulez&amp;utm_medium=propagace&amp;utm_campaign=labrulez_clanky_HPLCsystemy">Watrex Praha</a> on their social media (<a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/company/watrex/?viewAsMember=true">LinkedIn</a> and <a target="_blank" rel="noopener noreferrer" href="https://www.facebook.com/WatrexPrague">Facebook</a>) for more engaging topics.</blockquote>

Food Adulteration and How It Is Revealed by Analytical Chemistry

Join us for a fascinating conversation with Dr. Subhoja Chakraborty from the University of Central Florida, whose cutting-edge research targets the global challenge of malaria drug resistance. Dr. Chakraborty shares her journey from structure-based drug design during her PhD to her recent collaborative work repurposing human kinase inhibitors as rapid, selective antimalarials against drug-resistant Plasmodium falciparum strains.<h4>In this episode, you'll learn:</h4><ul><li>How structure-guided approaches reveal potential drug targets, including cysteine proteases like falcipain-2, and kinases implicated in malaria's complex life cycle.</li><li>The power of repurposing kinase inhibitors originally developed for cancer and other diseases, focusing on compound 12, which blocks hemozoin formation and protein kinase 6—key steps in parasite survival.</li><li>Lab techniques such as affinity, gel filtration, and size exclusion chromatography for recombinant protein purification—and how troubleshooting is crucial for high-yield, stable complexes.</li><li>Advances in mass spectrometry for characterizing protein interactions, validating drug targets, and mapping molecular pathways pivotal to malaria therapeutics.</li><li>Tips and lessons for new researchers facing the challenges of sample preparation, complex protocols, and working as part of a collaborative team.</li></ul>Dr. Chakraborty’s insights bridge the gap between basic science and translational drug discovery, offering hope for more effective, targeted malaria treatments in a world where resistance to frontline therapies threatens millions.<h4>Referenced Papers:</h4><ul><li>"Plasmodium falciparum protein kinase 6 and hemozoin formation are inhibited by a type II human kinase inhibitor exhibiting antimalarial activity" (Cell Chemical Biology, 2025)</li><li>"New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain" (BBRC, 2022)</li><li>"Structure-Based Optimization of Protease−Inhibitor Interactions to Enhance Specificity of Human Stefin‑A against Falcipain‑2 from the Plasmodium falciparum 3D7 Strain" (Biochemistry, 2023)</li></ul>If you’re a student, scientist, or simply passionate about advances in infectious disease research, this episode offers actionable insights, inspiring advice, and a front-row seat to innovation in combating malaria.<h3>Video Transcription</h3><h4>From Structural Biology to Drug Discovery: Chromatography and Mass Spectrometry in Malaria Research</h4><h5>Introduction</h5>In this discussion, Subhoja shares insights into her research journey, focusing on malaria drug discovery and the role of modern analytical techniques in biological and biomedical research. The conversation is aimed primarily at undergraduate students and early-career researchers interested in chemistry, biotechnology, and life sciences.<h5>Research Focus: Malaria Drug Discovery and Kinase Targets</h5>Subhoja is currently involved in malaria drug discovery research at the University of Central Florida, where she has been working since beginning her postdoctoral position two years ago. Her earlier PhD research focused on structure-based drug design in malaria, using a combination of in silico approaches, recombinant protein expression, and X-ray crystallography to determine precise three-dimensional structures of target proteins and their inhibitors.This structural perspective provided a foundation for her current work, which centers on drug discovery through compound screening and repurposing. A recent publication from her laboratory explores the use of repurposed mammalian CDK-like kinase inhibitors as potential antimalarial agents. While such kinase inhibitors are well known in cancer and cardiovascular research, their application to malaria targets represents a novel and promising approach.Kinases are attractive drug targets because of their central role in cell signaling and growth pathways. By targeting specific kinases involved in different stages of the malaria parasite life cycle—whether asexual growth, gametocytogenesis, or differentiation—it may be possible to significantly inhibit parasite growth and progression.<h5>Chromatography in Protein Expression and Purification Workflows</h5>Chromatography plays a critical role throughout Subhoja’s research. Her experience spans from basic chromatography techniques during her early training to advanced protein purification workflows using affinity chromatography, gel filtration (size exclusion chromatography), and ion exchange chromatography.Recombinant proteins are routinely purified using affinity resins such as nickel or cobalt, followed by further purification to remove contaminants. Fast Protein Liquid Chromatography (FPLC) enables compact, stepwise purification workflows suitable for producing proteins for in vitro assays.A recurring challenge in this process is the expression of high-molecular-weight proteins, which often accumulate in insoluble fractions or inclusion bodies. Careful selection of expression vectors, host systems, and induction conditions is therefore essential to maximize yield and solubility.<h5>Integrating Size Exclusion Chromatography into the Workflow</h5>Size exclusion chromatography (SEC) is typically used after affinity purification to further improve protein purity. Following elution with imidazole or glutathione, desalting is required to prevent buffer components from interfering with downstream assays. This is often achieved using desalting columns such as G-25 or PD-10.To remove lower-molecular-weight contaminants, finer SEC media—such as Superdex or Sephacryl columns—are employed. Concentration steps using molecular-weight cut-off filters are then applied to obtain the desired protein concentration for functional studies.These iterative purification and troubleshooting steps are a routine part of protein biochemistry and reflect the importance of methodical optimization and careful validation.<h5>The Role of Mass Spectrometry in Biomolecular Characterization</h5>Mass spectrometry is a key analytical tool in Subhoja’s research pipeline, particularly for target identification and validation. In both malaria and cancer research, MS is used alongside chromatography, immunoprecipitation, and pull-down assays to study protein interactions and molecular recognition events.Affinity-based mass spectrometry enables the identification of binding partners and interactors, whether studying proteins, long non-coding RNAs, or protein complexes. Techniques such as native MS, tandem affinity purification, and proximity labeling (e.g., TurboID-based workflows) provide deeper insights into molecular networks and signaling pathways.These approaches are essential for understanding how candidate drug targets function within complex biological systems.<h5>Sample Preparation Challenges in Biological Research</h5>Sample preparation remains one of the most challenging aspects of biological experimentation. Whole-cell lysates contain proteases, nucleases, and other components that can rapidly degrade target molecules. Preventing degradation requires minimizing preparation time, maintaining low temperatures, and using appropriate inhibitor cocktails.Reproducibility is also critical: biological experiments require multiple replicates, and variability is often unavoidable. Careful handling, protocol optimization, and continuous troubleshooting are essential to ensure reliable results, whether preparing samples for chromatography, mass spectrometry, sequencing, or immunoprecipitation.<h5>Advances in Chromatography and MS for Malaria Research</h5>Modern advances in chromatography and mass spectrometry significantly enhance malaria research by enabling precise target identification, quantification, and functional characterization. Drug discovery workflows begin with construct design and recombinant expression, followed by purification and interaction studies.In malaria research, stage-specific parasite biology requires careful extraction and analysis of proteins from different life cycle stages. Tandem affinity MS, immunoprecipitation, and in vivo validation studies help confirm target relevance and therapeutic potential.Recent work on repurposed kinase inhibitors has shown promising results, including inhibition of hemozoin formation—a key detoxification pathway for the malaria parasite—highlighting the potential of such compounds for further development.<h5>Malaria, Drug Resistance, and Global Impact</h5>Malaria remains a major global health burden, with over 600,000 deaths annually and widespread drug resistance complicating treatment. Artemisinin resistance, particularly associated with mutations in the K13 propeller domain, has driven the search for new targets and therapies.During her PhD, Subhoja focused on falcipain-2, a cysteine protease involved in hemoglobin degradation within the parasite. This pathway is critical for parasite survival and has emerged as a promising drug target. Her work revealed that falcipain-2 operates within a larger protein complex responsible for hemoglobin processing and amino acid release.Understanding these molecular mechanisms is essential for developing robust, stage-specific antimalarial strategies.<h5>Advice for Early-Career Researchers</h5>For researchers just starting out, Subhoja emphasizes the importance of hands-on experience. Working directly with cells, lysates, and analytical tools is essential for truly understanding experimental systems. Failure is an inherent part of research, and perseverance is key.Motivation, teamwork, careful sample handling, and thorough preparation all contribute to long-term success. Reading extensively, critically evaluating one’s own work, and appreciating incremental progress are vital aspects of a sustainable research career.Every successful experiment—no matter how small—represents meaningful progress and contributes to the broader goals of science and medicine.<h5>Closing Remarks</h5>The discussion highlights how interdisciplinary approaches combining chromatography, mass spectrometry, structural biology, and biochemistry are driving advances in malaria drug discovery. Through careful experimental design, analytical rigor, and collaborative teamwork, researchers can address complex biological challenges and contribute to global health solutions.<blockquote>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</blockquote><blockquote><h4>Concentrating on Chromatography Podcast</h4>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!You can find Concentrating on Chromatography Podcast in podcast apps:<ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL">Spotify</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185">Apple podcast</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography">podscan</a></li></ul>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos">YouTube channel</a></blockquote>

Affinity, Gel Filtration, Size Exclusion Chromatography: Repurposing Kinase Inhibitors for Malaria

Article

Product

Video

Scientists

Food

Interview

Science and research

The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences (IOCB Prague) is a leading scientific institution in the Czech Republic, recognized internationally. Its primary mission is basic research in the fields of chemical biology and medicinal chemistry, organic and material oriented chemistry, chemistry of natural compounds, biochemistry and molecular biology, physical chemistry, theoretical chemistry, and analytical chemistry. 

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences

Organomation, founded in 1959, designs and manufactures high-quality nitrogen evaporators and extraction systems. Known for innovation and durability, their lab instruments are used globally for efficient sample preparation with strong customer support.

Organomation

WATREX Prague is the distributor and producer of HPLC techniques in the Czech Republic and Slovakia. Main Chromatografic and Related Techniques Supported: LC, GPC, IC, IEC, Automated SPE, LC Columns and special column packings, LC method development, Software development, Laboratory Robots.

Watrex Praha

Agilent Technologies

Wiley

High molecular weight (HMW) aggregates and low molecular weight (LMW) fragments of biotherapeutic proteins induced by various stress conditions can be formed during the process of drug development, storage, shipment, or delivery. These size variants are critical quality attributes (CQAs) that need to be well characterized.
Size exclusion chromatography (SEC) HPLC is a commonly used technique to characterize and quantify size variants of these biotherapeutic proteins. The United States Pharmacopeia monograph method also recommends SEC for determining purity and aggregates of protein therapeutics.
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Presenter: Suresh Babu C.V. (Solution Development Scientist, Agilent Technologies)
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Dr. Suresh Babu C.V. completed his PhD in Biomolecular Science from University of Science and Technology (Korea Institute of science and Technology), South Korea and did his Postdoctoral fellow at School of Medicine, University of Cincinnati, USA. He has presented various papers in international and national conferences, and has published more than 25 papers in reputed peer reviewed journals. He has an extensive experience in biopharmaceutical characterization, method development, validation and in interpretation of chromatogram, electropherogram and mass spectral data for biomolecule analysis. Currently he is working as Solution Development Scientist at Agilent Technologies for past 10 years focusing on biopharmaceutical characterization. His areas of interest in Biopharma include- LC, LC/MS, CE, CE-MS, electrophoresis, and mass spectrometry.
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Presenter: Ansuman Mahato (Application Specialist LC, Agilent Technologies)
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Mr. Ansuman Mahato received his Master’s degree from Bangalore University in Biotechnology. He has been working in Agilent as senior application specialist for liquid chromatography in Center of Excellence lab. He carries rich experience on liquid chromatography platforms like GPC/SEC, HPLC, UHPLC, regulatory method transfers, life cycle management of analytical methods, and drive industry collaboration for new solution development. He has been developing applications for various market segments like Pharma, Food, chemical &amp; energy and biotherapeutics workflows.
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Presenter: Ms. Paramjeet Khandpur (Product Specialist, Agilent Techologies, India)
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Ms. Paramjeet Khandpur, M.Tech. received Bachelor’ degree in Chemical Engineering from VTU University and Master’s under Dr. Parag Gogte in Bioprocess Technology from the Institute of Chemical technology, Mumbai. During her research tenure, she has published 6 research articles in several international journals. Paramjeet is working as Application Scientist in Centre of Excellence Laboratory, Bangalore, Agilent Technologies India Pvt. Ltd. At Agilent, she has been working on liquid chromatography platforms like HPLC, UHPLC, 2D LC and preparative LC solutions. She is working in collaboration with several users in the Pharma/Biopharma industry for method development activities, HPLC to UHPLC method transfer, troubleshooting method related issues and has experience on various workflow of Pharma &amp; Bio-Pharma markets. Prior to joining Agilent, she worked as a faculty at Biotechnology Department, VTU university, Karnataka.
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Agilent Technologies: High Resolution Size Exclusion Chromatography (SEC) Analysis of Intact Protein Biotherapeutics

High Resolution Size Exclusion Chromatography (SEC) Analysis of Intact Protein Biotherapeutics

<h3>Application of 2D-LC-Q-TOF in the Structural Elucidation of Small Molecular Impurities during the Analysis of Active Pharmaceutical Ingredients</h3>
Two-dimensional liquid chromatography (2D-LC) has become well-known for its capability of separating complex mixtures. High resolving power and efficiency are the two most significant characteristics of 2D-LC. In the past decade, there is a growing interest in applying this efficient technology in the pharmaceutical industry, including the area of small synthetic molecules analysis. Special attention has been paid to complications that 1D-LC could not solve, such as the “hidden” or “non-resolved” peaks during the analysis of active pharmaceutical ingredients, the use of MS incompatible solvents during the LC-MS analysis, etc. In this presentation, the application of the Agilent 2D-LC-Q-TOF in the structural elucidation of impurities of small molecules will be discussed through several case studies.
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Presenter: Ivan Dai, PhD (Senior Scientist, Takeda)
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Ivan Dai earned his Bachelor of Science in Food Science from China Agricultural University in 2008. After earning his Master of Science in Food Science from Virginia Tech, he received his Ph.D. in Natural Products and Medicinal Chemistry in 2013, working with Professor David Kingston. Following completion of his graduate studies, he spent an additional three years at Virginia Tech doing post-doctoral training in the area of enzymology and chemical biology. After working briefly as a senior research associate in a biopharma Continuum Biosciences, he joined the Structural Characterization group in Analytical Development at Takeda Pharmaceuticals in Cambridge, MA in 2019. He is currently a Senior Scientist at Takeda with research interests involving structural elucidation of impurities as well as leachables and extractables generated during API process by LC-MS, GC-MS, NMR, together with other analytical methods, including novel techniques (2D-LC-Q-TOF, RDC measurement, forced degradation etc.). In his spare time, Ivan also serves as a topic editor for the journal Biomolecules (MDPI).
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<h3>Algorithms and Workflows for Identification of Impurities and Metabolites</h3>
Automated algorithms and workflows for the analysis of small molecule impurities and metabolites will be discussed. High resolution accurate mass (HRAMS) results are necessary for the use of these algorithms. The two main types of HRAMS analyzers will be briefly compared and contrasted. The use of mass defect, common reporter or fragment ions, and common neutral loss ions in an automated workflow are reviewed. Methodology for maximizing duty cycle such as preferred lists, exclude lists, and mass defect directed MSMS are also described. The characterization of unknown candidates via library matching and database matching are important parts of the workflow. Agilent library matching and MSC structure elucidation along with open source structure elucidations like CFM-ID and Sirius will also be discussed.
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Presenter: Jim Lau, PhD (HRAMS LC/MS Application Engineer, Agilent Technologies, Inc.)
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Jim Lau received his Ph.D. From the University of Colorado. There he worked in the laboratories of Peter Albersheim and Robert Shapiro. This work involved the structure elucidation of plant natural products and, in particular, elucidating the structures of oligosaccharides derived from plant cell wall polysaccharides. This work was done in the early days of LCMS and made use of DLI-LCMS and (at the time) recently invented Thermospray-LCMS.
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After the completion of his Ph.D., Jim joined Hewlett-Packard to work on LCMS ionization techniques with Paul Goodley during the early days of Electrospray-LCMS. He then moved to work on field applications and collaborations with Hewlett-Packard’s LCMS customers. More than 30 years later, Jim continues to work with Agilent’s customers in High Resolution Accurate Mass applications of LCMS. While involved in many areas of HRAMS LCMS, his recent work has focused on the area of Screening/Qual&amp;Quant of small molecules and on the high throughput confirmation of oligonucleotidesynthesis.
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Agilent Technologies: Experts Webinar Series: Small Molecule Pharma

Experts Webinar Series: Small Molecule Pharma

Adeno-Associated Viruses (AAVs) are the main viral vectors for gene therapy. Intact AAVs act as a vehicle to protect and deliver therapeutic oligonucleotides. As part of critical quality attributes (CQA) of therapeutic products, analytical methods such as Ion Exchange Chromatography (IEX) and Liquid Chromatography Mass Spectrometry (LC-MS) were used for the separation of AAV capsids and characterization of reduced subunits.
This seminar will describe the applicability of Two-Dimensional Liquid Chromatography (2DLC) coupled with Mass Spectrometry to automate the two orthogonal techniques of IEX and LC-MS in the characterization of Empty and Full AAV Capsids.
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Presenter: Dat Phan (Applications Scientist, Agilent Technologies, Inc.)
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Dat Phan is an Applications Scientist in the Liquid Phase Separations Group at Agilent Technologies. Before joining Agilent Technologies (HP at that time) in 1988 Dat was with the University of Missouri-Columbia where he received his academic and professional trainings in Chemical Engineering and Analytical Biochemistry. Dat supports ChemStation Software and applications in HPLC, UHPLC, LCMS, CE, and CEMS.
</blockquote>

Agilent Technologies: 2DLC-MS Applicability for the Characterization of Adeno-Associated Viruses

2DLC-MS Applicability for the Characterization of Adeno-Associated Viruses

<h3>Summary</h3>
<h5>Recent Advances in 2D-LC for Biopharmaceutical Analysis - Fundamental Studies and Practical Applications</h5>
Characterization of biomolecule (e.g., peptides, proteins, oligonucleotides) mixtures is typically quite challenging due to the heterogeneity of the molecules themselves (e.g., due to extensive glycosylation), and/or the matrices in which they are presented to the analytes (e.g., cell culture supernatant). Increasingly, two-dimensional liquid chromatography (2D-LC) methods are being used to improve the depth of characterization of these mixtures, improve the speed of these analyses, or both. This seminar will provide an overview of the key concepts required for successful 2D-LC method development, and several examples of recently developed 2D-LC separations of biomolecules.
<h5>Learning Objectives:</h5>
<ul>
<li>Learn about innovative ways 2D-LC is being used to characterize molecules ranging from oligonucleotides to proteins </li>
<li>Learn about the most recent fundamental studies that support development of better 2D-LC methods </li>
<li>Learn about the trend in the use of multi-dimensional liquid chromatography to automate deep characterization of mAbs and other proteins</li>
</ul>
<blockquote>
Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)
</blockquote>
<blockquote>
Dwight Stoll is Professor of Chemistry at Gustavus Adolphus College in St. Peter, MN. He has authored or co-authored 74 peer-reviewed publications and four book chapters in separation science, and over 130 conference presentations. His primary research focus is on the development of two-dimensional liquid chromatography (2D-LC) for both targeted and untargeted analyses. Within this area he has made contributions on many aspects of the technique including stationary phase characterization, biopharmaceutical analysis, new 2D-LC methodologies and instrumentation, and fundamental aspects including re-equilibration in gradient elution reversed-phase liquid chromatography and analyte focusing. He has taught several short courses on 2D-LC in recent years at venues including Pittcon and the international HPLC 20XX series. He is the 2011 recipient of LCGC’s Emerging Leader in Chromatography Award, the 2015 recipient of the American Chemical Society Division of Analytical Chemistry Award for Young Investigators in Separation Science, and in 2017 he received the Georges Guiochon Faculty Fellowship.
</blockquote>
<h5>Two-Dimensional Liquid Chromatography (2D-LC): A Tutorial focusing on Best Practices - Part 1</h5>
Protein biologics such as mAbs now represent a significant share of pharmaceutical sales and future growth potential, particularly in an era of increasing patent expirations. LC/MS is a powerful analytical method for characterizing therapeutic proteins. In this presentation, we will show the application of high resolution and high mass accuracy Q-TOF for intact mAb measurement, peptide mapping, and characterization of PTM (Post-Translation Modifications). Fast separation using AdvanceBio peptide mapping columns with a state-of-the-art HPLC system will also be highlighted.
<h5>Learning Objectives:</h5>
<ul>
<li>Learn about different modes of 2D-LC and how these are enabled by recent advances in instrumentation </li>
<li>Learn about column (stationary phase and dimensions) selection and optimizing usage of available 2D separation space</li>
</ul>
<blockquote>
Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)
</blockquote>
<h5>Two-Dimensional Liquid Chromatography (2D-LC): A Tutorial focusing on Best Practices - Part 2</h5>
Monoclonal antibodies (mAb) are glycoproteins of the immunoglobulin (Ig) family. They can be used as therapeutics for the treatment of several life-threatening conditions such as cancer, inflammatory diseases, complications arising after organ transplantation, infectious, and cardiovascular diseases. Like all other protein therapeutics, mAbs can undergo aggregation during production, storage and transportation. Aggregation of therapeutic proteins is undesirable since it can lead to activity loss, decreased solubility, and enhanced immunogenicity. Usually, run under isocratic conditions, SEC method development is fairly straightforward. However, many analysts do not fully explore how changes in system volume effect sizing chromatography. Discover more about the new 1290 Bio LC and learn how minimizing dispersion volume improves resolution.
<h5>Learning Objectives:</h5>
<ul>
<li>Learn about the mobile phase mismatch problem, and different strategies to minimize its effects </li>
<li>Learn about important method development fundamentals, including loop filling and interface operation </li>
<li>Learn about strategies for approaching developing 2D-LC methods</li>
</ul>
<blockquote>
Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)
</blockquote>
<h5>Ask the Experts</h5>
Still have 2D-LC questions? Regardless of whether they are hardware, software or application based, bring your questions and ask our expert panelists!
<blockquote>
Experts: Dwight Stoll, Stephan Buckenmair, Ulrich Ederhardinger
</blockquote>
<blockquote>
Presenter: Dr. Stephan Buckenmaier (Principal Scientist, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Stephan has received his PhD from the University of the West of England (Bristol, UK) in 2004, where he investigated “Factors influencing Retention and Peak Shape in HPLC” in the group of Prof. David McCalley. Following a Postdoc at the Proteome Center at the University of Tübingen, where he studied diagnostic proteins and post-translational modifications, he joined Agilent in 2006. Currently, Stephan holds a position as Principal Scientist in Research and Development in the Liquid Phase Separation Division located in Waldbronn, Germany. His primary focus is on the development of 2D-LC workflow solutions and collaborations with external companies and academic institutions.
</blockquote>
<blockquote>
Presenter: Dr. Ulrich Eberhardinger (Product Manager, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Ulrich studied chemistry at the University of Stuttgart and earned his PhD in physical chemistry. He works for Agilent since 2002 and has been responsible for the development and market introduction of various hardware, software products and solutions in liquid chromatography and microfluidics such as the bio-inert LC, 1260 and 1290 Infinity II systems, high-performance pumps, method development solution, instrument control software and many others. As the product manager for 2D-LC Software, he shapes the future of multidimensional chromatography at Agilent.
</blockquote>

Agilent Technologies: 2D-LC Virtual Forum

2D or Not 2D - Agilent 2D-LC Forum

During the manufacturing process, protein therapeutics (mAbs) are subjected to several posttranslational modifications (PTMs) which will affect the biochemical and biophysical properties of the mAbs. One of such events is charge variants and its determination is one of the most important quality attributes for the characterization of biopharmaceuticals such as monoclonal antibodies (mAbs). Traditionally, charge variant profiling is done using ion exchange chromatography (IEX), fractionation collection, followed offline desalting and LC-MS analysis to qualify the charge variant pattern of the mAbs. This approach is not a straightforward procedure, mostly due to the incompatibility of the IEX buffers (phosphate buffers containing sodium chloride) with the electrospray ionization process.
However, a 2D-LC solution enables automated desalting, and offers additional separation by the reversed-phase chromatography (RP) in the second dimension.
In this webinar, the speakers will discuss the importance of charge variants analysis of therapeutic mAbs and a fully automated multiple heart-cutting 2D-LC/MS workflow solution for charge variant characterization.
<blockquote>
Presenter: Prof. Anurag S. Rathore (Department of Chemical Engineering, Indian Institute of Technology, Delhi)
</blockquote>
<blockquote>
Presenter: Ms. Paramjeet Khandpur (Product Specialist, Agilent Techologies, India)
</blockquote>
<blockquote>
Presenter: Dr. Ashish Pargaonkar (Senior Applications Scientist, Agilent Technologies, India)
</blockquote>

Agilent Technologies - BioPharma webinar series

Fully automated 2D-LC/MS for biotherapeutics charge variant characterization using Ion Exchange (IEX) and Reversed-Phase (RP) Chromatography

Pitfalls in the coupling of bioanalytical separation techniques and mass spectrometry, as well as their elimination, will be explained.
<h3>Who should attend:</h3>
<ul>
<li>Bioanalysts, pharmacists, instrumental analysts, quality inspectors, users of 2D-HPLC.</li>
</ul>
<blockquote>
Presenter: Lars M. H. Reinders (Scientist, Institute for Energy- and Environmental Technology e. V. (IUTA))
</blockquote>
<blockquote>
Lars M. H. Reinders completed his studies in applied chemistry with a focus on instrumental analysis at University of Applied Sciences, Krefeld in 2017. Afterwards, he started his PhD at the Faculty of Chemistry at the Chair of &quot;Instrumental Analytical Chemistry&quot; at the University of Duisburg-Essen. His research focus is on analytical support of aseptic preparation, where he develops and validates analytical methods for quantification and identification of monoclonal antibodies.
</blockquote>

Wiley Analytical Science: Characterisation and Quantification of Biomolecules

Characterisation and Quantification of Biomolecules

Finally, an outlook on current research topics is given, which among other things focus on new and innovative stationary phases based on percellulose.
The analysis of properties of monoclonal antibodies (MAbs) and related products (e. g. ADCs) relies on various liquid chromatographic modes based on different separation mechanisms. While SEC distinguishes the size variants such as aggregates and fragments, IEX separates acidic and basic charge variants. RP analyses proteins mainly according to their hydrophobicity and HIC is the standard to obtain relative hydrophobicities and the drug to antibody ratio of antibody-drug-conjugates. 
This web seminar gives you a comprehensive overview about LC techniques designed for MAbs in SEC, IEX, RP, and HIC using state-of-the-art (U)HPLC columns. In addition, you will learn about special approaches such as direct coupling of IEX-MS or HIC-MS.
<h3>Who should attend:</h3>
This web seminar is dedicated for all kind of BioLC users, whether advanced ones or beginners, who want to get input on solutions based on different LC modes.
<h3>In this web seminar you learn:</h3>
<ul>
<li>Separation modes applicable for Mab analyses.</li>
<li>What to keep in mind using different LC modes.</li>
<li>Selection of the best suitable columns according to the separation task.</li>
<li>Ways to speed up your analyses.</li>
<li>Options for direct coupling of RP, IEX and HIC to MS.</li>
</ul>
<blockquote>
Presenter: Dr. Daniel Eßer (Product Manager Analytical Chromatography, YMC Europe GmbH, Dinslaken, Germany)
</blockquote>
<blockquote>
Daniel Eßer studied Chemistry at the Applied University Bonn-Rhein-Sieg, in Rheinbach with the focus on pharmaceutical and analytical chemistry. He received his PhD in Pharmaceutical and Medicinal Chemistry at the University of Düsseldorf. During his Postdoc at the Institute of Pharmaceutical and Medicinal Chemistry of the University of Düsseldorf, he establised a nanoLC-MS system. In 2013 he joined YMC Europe as Product Specialist Analytical Chromatography. Since 2017, he is responsible for the analytical (U)HPLC column portfolio as Product Manager Analytical Chromatography. 
</blockquote>

Wiley Analytical Science: Analysing Antibodies by Different LC Separation Modes

Analysing Antibodies by Different LC Separation Modes

2D HPLC is the selective transfer of a fraction or fractions from one column to another for further separation. It can be used in a variety of situations including:
<ul>
<li>Further resolution of a complex mixture that can’t be separated on a single column (1D chromatography)</li>
<li>Sample cleanup, removing matrix or interfering compounds</li>
<li>Trace enrichment</li>
</ul>
While using reversed phase in both dimensions is the most common combination for 2D HPLC, it has the lowest orthogonality. We’ll look at how combining orthogonal techniques, like IEX and RP can help you achieve optimum resolution and discuss the advantages and disadvantages of these combinations.
<blockquote>
Presenter: Rita Steed (Application Engineer, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Rita Steed began supporting the LC column line for Agilent in 1999. As a Chromatography Specialist (first with Chromatography, Inc., then directly for Agilent), Rita worked on-site with researchers in Pharmaceutical and other industries presenting technical seminars and assisting researchers with troubleshooting and method development. Rita has over 20 years of Chromatography experience in the Biotechnology, Chemical, and Pharmaceutical industries. She has held positions in Research, Sales, and Technical Service. Rita has earned degrees in Microbiology and Life Sciences/Biochemistry. In her current position, she is an inside Application Engineer supporting LC columns.
</blockquote>

LCMS webinars focusing on 2D-LC - page 3

Is 2D HPLC the Answer for You?