GenNext Open Access Video Resource Library
mAb Characterization Using Flash Oxidation Technology and Mass Spectrometry
Deepen your knowledge of protein High Order Structure (HOS) for therapeutics with protein footprinting technology paired with high-resolution mass spectrometry, using both CID and EAD peptide fragmentation. HOS is crucial for a drug’s stability, safety, and function, and misconfigurations can cause adverse reactions. Hydroxyl Radical Protein Footprinting (HRPF) with the Fox® System helps explore factors like epitope mapping, aggregation, formulation, and small molecule binding, enhancing therapeutic HOS understanding.
mAB Therapeutic Characterization using Fox Protein Footprinting
Using the Fox® System, researchers have advanced the understanding of therapeutic HOS by exploring epitope/paratope mapping, aggregation-interface identification, formulation, and small molecule binding. The Fox System, a novel HRPF technique, employs hydroxyl radicals (•OH) to irreversibly modify solvent-exposed amino acid side chains, revealing changes in solvent accessibility and uncovering buried or exposed regions. This presentation will discuss the Fox System, its application in characterizing biotherapeutic HOS, and future innovations.
Fox System Elucidates Changes in Peptide to Amino Acid Solvent Accessibility
The Fox® Protein Footprinting System uses hydroxyl radical protein footprinting (HRPF) by photolyzing H2O2 with a plasma flash lamp. It includes fluidics, photolysis, dosimeter, and product collector modules. The fluidics module transports samples and H2O2, while the photolysis module generates •OH radicals to label proteins. The dosimeter measures •OH load by detecting changes in absorbance. Labeled samples are collected in quench solution, digested, and analyzed by LC-MS/MS. FoxWare® Software identifies and visualizes significant oxidation changes, mapping them to protein models.
Fox HRPF for mAb Drug Development
The Fox® Protein Footprinting System uses hydroxyl radical protein footprinting (HRPF) with a proprietary flash oxidation lamp to generate hydroxyl radicals (•OH) from hydrogen peroxide. This modifies solvent-exposed amino acids, reflecting changes in solvent accessibility. HRPF with LC-MS/MS quantifies these modifications at peptide and amino acid levels. The system identified interaction surfaces of TNFα and Adalimumab by adjusting flash lamp voltage to maintain consistent •OH concentrations. FoxWare® Software analyzed tryptic digestion and proteomics data, revealing decreased oxidation in regions corresponding to the paratope or epitope in the known crystal structure, indicating protection from •OH modification. The Fox System effectively maps antibody-antigen interfaces.
Fox System Accurately Maps the Paratope and Epitope of TNFα Bound to Adalimumab
A study on TNF alpha and its monoclonal antibody adalimumab, using the Fox Protein Footprinting System, revealed oxidation changes in TNF alpha peptides matching the known epitope. Adalimumab’s Fab region showed decreased oxidation due to reduced solvent accessibility when bound to TNF alpha, while the Fc region had increased oxidation, indicating flexibility beyond the crystal structure. This method accurately mapped the TNF alpha epitope and adalimumab paratope, providing insights beyond the crystal structure.
Fox HRPF for mAb Aggregation
In under 10 minutes, Dr. Chea explains how the Flash Oxidation (Fox®) Protein Footprinting System can characterize monoclonal antibody (mAb) aggregation and assess how excipients stabilize proteins. Excipients, such as amino acids, help limit aggregation. By comparing hydroxyl radical modifications before and after amino acid addition, you can detect intermolecular interactions during mAb aggregation. Understanding the higher order structure of protein aggregation equips you with the insights needed to optimize mAb formulation and development.
THE SCIENCE OF HRPF
FPOP for Problem Solving in Protein Chemistry and Biology
FPOP has become a versatile tool in protein chemistry, offering microsecond-speed footprinting to track rapid protein folding and conformational changes, even those missed by slower techniques. It aids in epitope mapping, detects conformational changes in biopharmaceuticals, and studies protein-ligand interactions relevant to diseases like Alzheimer’s. FPOP is effective for membrane proteins and in living cells, providing unique insights. The lecture will compare FPOP with other footprinting methods.
The Fox System: A Novel, Laser-Free POP Footprinting Platform
Dr. Sharp discusses a covalent labeling technique called fast photochemical oxidation of proteins (FPOP), detailing the workflow, its application in research, and how a new platform is designed to enhance user-friendliness. The Fox Oxidation System streamlines the entire process, from radical generation to sample collection, improving safety, reliability, and ease of use compared to traditional FPOP setups. This innovation makes HRPF accessible to more researchers, allowing them to gain valuable insights into protein structures and interactions with greater ease.
Protein Footprinting Applications in Structural Biology
This hour-long seminar, hosted with The Analytical Scientist, reviews structural biology case studies showcasing the successful application of protein footprinting in solving key problems in biopharmaceutical research. Examples include epitope and paratope mapping, protein interactions, receptor-drug interactions, and protein folding/unfolding. The seminar highlights the benefits of using protein footprinting technology in advancing biopharmaceutical research.
Introduction to Protein Footprinting
This 60-minute webinar, in collaboration with Cambridge Healthtech Institute, offers a comprehensive review and instruction in protein footprinting techniques and applications. Learn about various benchtop covalent modification methods and rapid labeling techniques like X-ray synchrotron and Fast Photo-Oxidation of Protein (FPOP) hydroxyl radical protein footprinting (HRPF). Explore their practical implications for biopharmaceutical research, protein interactions, biosimilar development, epitope and paratope mapping, ligand-binding, and conformational change analysis.
Fast Photochemical Oxidation of Proteins (FPOP) HRPF
This 60-minute webinar offers in-depth instruction on the theory, experimental design, and practical execution of FPOP HRPF experiments. The focus will be on achieving robust and reproducible labeling through real-time radical dosimetry, with a strong emphasis on exemplary applications.
HRPF Data Processing and Higher Order Structural Analysis
This presentation provides a detailed discussion on data processing and analytics for HRPF studies. It emphasizes effective methods for peptide and residue-level differential analysis and explores the practical implications of changes in protein higher-order structure due to ligand binding, antibody-antigen interactions, and protein interaction dynamics.
APPROACHES TO STRUCTURAL BIOLOGY
Hacking Structural Biology for Drug Discovery Using Mass Spectrometry
Mark Chance discussed his vision for proteomics and structural biology, focusing on megadalton protein complexes and the technologies needed to study them. He highlighted the importance of combining techniques like NMR, crystallography, cryo-EM, and mass spectrometry for protein interaction insights. He explained hydroxyl radical footprinting, a method for mapping protein surfaces and how it has advanced drug development. He shared examples from Rodeo Therapeutics, CASMA, and Foghorn Therapeutics, showcasing its practical applications.
HRPF and its Relevance in Drug Development
This presentation reviews technology options for studying protein high order structure (HOS) in drug development. Dr. Chea categorizes techniques by ease of use, information value, and cost. Low-resolution methods are affordable but limited in insights, while high-resolution techniques like X-ray crystallography, cryo-EM, and NMR offer atomic detail but are more complex and costly. Mass spectrometry-based methods, such as chemical cross-linking, HDX, and HRPF, balance usability with valuable information, making them essential for protein analysis in research and drug development.
Technologies for Higher Order Structural Analysis
Many structural biology researchers seek easier and more robust methods for Higher Order Structural (HOS) analysis, recognizing its critical role in biotherapeutic stability, safety, and function. Traditional HOS approaches can be complex, slow, dangerous, and costly, or unreliable and uninformative. With many factors to consider, choosing the best method for your lab can be challenging. Dr. Chea reviews various options, highlighting their pros and cons, and introduces the Flash Oxidation (Fox®) Protein Footprinting System. She explains its applications in epitope mapping, mAb aggregation, and conformational analysis, helping you select the best method for developing safe and effective biotherapeutics.
EMERGING APPLICATIONS OF HRPF
Radical Protein Footprinting in Mammalian Whole Blood
Radical protein footprinting (RPF) is a structural biology technique used in cells and organisms. This presentation describes the first example of radical protein footprinting in mammalian blood using sulfate radicals. It shows the first oxidation of proteins by sodium persulfate using a Fox Protein Footprinting System, and the suitability of adenine dosimetry for measuring sulfate radical dose. The research also introduces improved mixing and quenching methods to reduce background oxidation, successfully applying RPF to stabilized whole mouse blood.
Broadening the Scope of Hydroxyl Radical Protein Footprinting Applications
Professor Sharp discusses advancements in protein footprinting technology, including the ability to perform experiments on benchtops using UV-catalyzed hydroxyl radicals for protein labeling. He explains how this technique can address challenges in studying dynamic, heterogeneous systems like glycoproteins, aggregation processes, and phosphorylation. Applications include studying HIV glycoproteins, malaria proteins, and monoclonal antibody aggregation.
Moving to in-cell and in-vivo Applications
In-cell and in vivo hydroxyl radical protein footprinting (FPOP) enables studying protein structure and interactions directly within cells or live organisms, crucial for understanding molecular crowding’s effects. The method, using UV lasers to split hydrogen peroxide, labels proteins in situ. Recent developments, including single-cell flow systems and the PIXI platform, increase throughput and enable analysis of complex models like 3D cultures.
In-cell and in vivo FPOP
This hour-long seminar reviews recent advancements in in-cell and whole organism FPOP. It offers a detailed discussion on experimental design and format, highlighting the strengths and benefits of these methods compared to in vitro FPOP. The seminar also covers the implications for protein HOS research and drug development, featuring real research examples from eukaryotic cells and C. elegans.