Analytical Techniques for Biopharmaceutical Development

This excellent paper by Evans et. al. describes studies of targeted CQA analytical control strategy for commercial antibody products: replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring. Quoting from the abstract: "Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities."

Ok, hear me out, are we in front of a game-changing platform for analyzing LNPs and their cargo? This new study introduces an innovative IPRP HPLC method with UV and CAD detection, enabling simultaneous quantification of lipids and nucleic acids in LNPs. This platform streamlines analytics for LNP-based nucleic acid therapeutics, tackling a key challenge in formulation development. Some key findings your way: 1) Versatile quantification - The method separates and quantifies 12 lipid species (e.g., cholesterol, SM-102, DSPC) and nucleic acids (ASO, sgRNA, mRNA) spanning 17 to ~4500 nucleotides, using a HALO 400 Å C4 column with optimized conditions (100 mM DBA, pH 6.8, IPA gradient). 2) Efficient recovery - A single-step sample prep with Triton X-100 and heparin achieves near-complete extraction of both lipids and nucleic acids (>87% recovery), even for complex co-loaded sgRNA/mRNA LNPs, with results matching orthogonal RiboGreen assays. 3) Process insights - Applied to in-process samples, it revealed consistent ASO recovery (~100%) post-centrifugal filtration, while lipid recovery varied (82-110%), highlighting potential losses from empty particles or membrane adsorption during purification. Couple of limitations tho -the method’s robustness was tested on a limited set of LNP formulations, and its applicability to highly diverse lipid libraries or extreme N/P ratios remains unverified. Long-term column stability under repeated use and scalability for high-throughput screening also need further exploration, in my opinion. Read the full study here: https://lnkd.in/ePkEPtuk #AnalyticalChemistry #LipidNanoparticles #mRNATherapeutics #Chromatography #DrugDelivery #BiotechInnovation #FormulationScience

𝗰𝗜𝗠𝗦 for 𝗔𝗱𝘃𝗮𝗻𝗰𝗲𝗱 𝗖𝗵𝗮𝗿𝗮𝗰𝘁𝗲𝗿𝗶𝘇𝗮𝘁𝗶𝗼𝗻 of #sgRNA 𝘞𝘩𝘢𝘵 𝘥𝘰 𝘺𝘰𝘶 𝘥𝘰 𝘰𝘯𝘤𝘦 𝘺𝘰𝘶 𝘮𝘦𝘢𝘴𝘶𝘳𝘦 𝘢𝘯 𝘪𝘯𝘵𝘢𝘤𝘵 #𝘊𝘙𝘐𝘚𝘗𝘙 𝘨𝘶𝘪𝘥𝘦 𝘙𝘕𝘈 𝘮𝘢𝘴𝘴? 𝘠𝘰𝘶 𝘤𝘩𝘢𝘳𝘢𝘤𝘵𝘦𝘳𝘪𝘻𝘦 𝘪𝘵𝘴 𝘤𝘰𝘮𝘱𝘰𝘴𝘪𝘵𝘪𝘰𝘯, 𝘰𝘧 𝘤𝘰𝘶𝘳𝘴𝘦... You can do this with nuclease-based oligo mapping (#RapiZyme RNases), and you can do this with gas phase fragmentation if you have the #HRMS capabilities to take on advanced techniques. 💡Regarding the latter, Luis Macias, Jamie Lowther, Eric T., Ellen Rohde, and James Madsen at Verve Therapeutics just published a super neat example! They paired 𝘁𝗼𝗽-𝗱𝗼𝘄𝗻 𝗠𝗦 𝘄𝗶𝘁𝗵 𝗰𝘆𝗰𝗹𝗶𝗰 𝗶𝗼𝗻 𝗺𝗼𝗯𝗶𝗹𝗶𝘁𝘆 (𝗰𝗜𝗠𝗦) to reach a breakthrough on sequence and modification mapping. What stands out in their work: 🔹They hit 95% sequence coverage on a synthetic gRNA loaded with 2'-O-methylations. 🔹Ion mobility resolved overlapping fragment ions and increased isotopic fidelity—greatly improving assignment confidence and the potential to apply de novo sequencing. 🔹Regions rich in methylations were comprehensively mapped at the same time that truncations and phosphorothioate impurities were identified. 💎 If you’re working on analytical methods for CRISPR therapeutics, this paper is a gem. 𝗢𝗽𝗲𝗻 𝗮𝗰𝗰𝗲𝘀𝘀:  https://lnkd.in/ecH8i6YN #AnalyticalDevelopment #CMC #AnalyticalChemistry #GeneTherapy #CellTherapy ---------------------------------- Follow for more content on analyzing biomolecules 🔔

Analyzing Heterogeneities in mRNA-Lipid Nanoparticles: Utilizing Sucrose Density Gradient Ultracentrifugation Rational design and robust formulation processes are essential for optimal mRNA delivery via lipid nanoparticles (LNPs). Heterogeneity in mRNA-LNPs can significantly impact their biophysical and functional properties. Given the complexity of mRNA-LNPs, developing comprehensive analytical techniques is crucial for better understanding these formulations. In this study, we present a robust ultracentrifugation method for density-based separation of mRNA-LNP subpopulations. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using different lipids. Upon ultracentrifugation on a sucrose gradient, distinct fractions were observed, ranging from less dense to denser fractions, which were analyzed comprehensively. Parent LNPs were resolved into density-based fractions, differing significantly in hEPO expression following administration routes. This study demonstrates the application of density gradient-based ultracentrifugation for a head-to-head comparison of heterogeneity and its impact on biological performance and biophysical characteristics of mRNA-LNPs and their subpopulations. #mRNA #LipidNanoparticles #DrugDelivery #Formulation #Heterogeneity #Ultracentrifugation #AnalyticalTechniques #BiophysicalProperties #BiologicalPerformance #DensityGradientCentrifugation APURUN https://lnkd.in/ge7GwfZk

Advanced Analytical Techniques for Lipidomics Advanced analytical techniques in lipidomics have revolutionized our understanding of lipid biology by enabling comprehensive analysis of lipid species and their biological functions. These techniques allow researchers to identify, quantify, and study the diversity and complexity of lipids in cells, tissues, and biofluids with high precision and sensitivity. The most prominent analytical methods include mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and chromatography-based techniques. (1) Mass spectrometry (MS): MS is the cornerstone of lipidomics due to its high sensitivity, accuracy, and ability to identify and quantify lipids on a large scale. Various MS-based methods, such as tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS), are widely used to analyze the structure of lipid molecules. MS provides detailed insights into lipid composition, including molecular weight and fragmentation patterns, and can even detect low-abundance lipid species. Combining MS with chromatographic techniques, such as liquid chromatography (LC-MS) and gas chromatography (GC-MS), can enhance the separation and identification of complex lipid mixtures. (2) Nuclear magnetic resonance (NMR) spectroscopy: NMR allows non-destructive analysis of lipids, providing information about lipid structure and dynamics in their native state. It is particularly useful for studying lipid-protein interactions and the behavior of lipids in cell membranes. Although not as sensitive as MS, NMR is very useful for quantifying lipids in mixtures and elucidating the functions of lipids in biological systems. (3) Chromatographic techniques: Chromatographic methods such as liquid chromatography (LC), gas chromatography (GC), and thin layer chromatography (TLC) are used to separate lipids based on their physicochemical properties. Used in conjunction with MS or other detectors, these techniques can separate complex lipid species, thereby enhancing lipid identification and quantification. Together, these advanced analytical techniques provide a comprehensive toolkit for lipidomics research, enabling the exploration of lipid metabolism, signaling pathways, and their role in health and disease. References [1] Kui Yang and Xianlin Han, Trends in Biochemical Sciences 2016; 41: 954-69 [2] Tianrun Xu et al., Anal Chim Acta 2020 (10.1016/j.aca.2020.09.060) #Lipidomics #MassSpectrometry #NMR #Chromatography #LCMS #GCMS #LipidAnalysis #Bioinformatics #LipidMetabolism #AdvancedTechniques #LipidProfiling #BiomedicalResearch