Applications, Challenges and Opportunities for Top-Down, Intact, and Native Mass Spectrometry in Biopharmaceutical Characterization

Applications, Challenges, and Opportunities for Top-Down, Intact, and Native Mass Spectrometry in Biopharmaceutical Characterization

Thursday, February 12, 2026, 11:00AM-2:00PM ET

With the increase in the development of new modalities as well as the expansion of the ability to characterize large molecules, there is a need for an in-depth assessment of the state of this arena. This Workshop will examine the current applications and opportunities as well as look at the future needs of the industry. The 8 speakers will present their latest work on the use of top-down, intact, and native mass spectrometry in characterizing a range of antibody-drug conjugates, fusion proteins, bi-/tri-specific monoclonal antibodies, antibody variants, adeno associated viruses, oligonucleotide therapeutics, and aggregate species.

This will be followed by a dynamic Panel Discussion where the speakers will share their perspectives on the current challenges, new opportunities, as well as future directions. The Workshop and Panel Discussion will be led by Jared Auclair of Northeastern University.

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Intact Mass for ADC BioAnalysis in Practice: PK, DAR, and Payload Measurements

John Kellie - AstraZeneca, Gaithersburg, Maryland

Bioanalytical strategies for pre-clinical ADC studies is often multi-faceted, including total antibody (TAb) and total ADC (TADC) quantitation (digestion approaches). Intact/reduced mass analyses of ADCs from in vivo samples provide a top-down view of ADC stability. Presented herein are current, cutting-edge MS strategies for pre-clinical ADC bioanalytical support. This includes aspects of traditional bioanalysis practices for large and small molecules, intact mass analysis approaches, details on hybrid immune-affinity MS formats, and payload quantitation.

State-of-the-art characterization of in vivo biotransformations of biotherapeutics on the new tims Omni platform

Lucile Kogey-Fuchs - Sanofi, Vitry-sur-Seine, France

An important step in the development of new biotherapeutics is assessing their in vivo stability and identifying biotransformations that may impact their efficacy and safety. A top-down MS workflow using the new timsOmni instrument was developed to enable proteoform-level characterization and recover structural information lost at the peptide level. Intact-mass profiling of plasma from mice administered a hybrid mAb (150 kDa) identified 12 low-abundance proteoforms and guided MS/MS analysis, enabling characterization of ~100 kDa species and providing structural insight into in vivo biotransformation pathways.

Proteoform Microheterogeneity Assessment of Antibody-based Biopharmaceuticals using Native Top-Down Mass Spectrometry

Corentin Beaumal - National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland

Native mass spectrometry, coupled with non-denaturing size-exclusion and pH-gradient ion-exchange chromatography, enabled detailed intact biopharmaceutical characterization under near-native conditions. When combined with Orbitrap MS, these separation formats enable confident identification of proteoforms and facilitate the evaluation of posttranslational modifications that may cause subtle changes in protein mass. Here, we performed LC-MS profiling of mAbs and bispecific antibodies under near-native conditions. Separation using pH gradient cation exchange chromatography combined with native top-down MS using multiple ion activation techniques (HCD, ETD, EThcD, and UVPD), and resulting spectra further simplified using proton-transfer charge reduction (PTCR), enabled intact mass determination and proteoform modifications identification for each of the monoclonal antibodies analysed, with complex profiles in both acidic and basic regions identified. Overall, the high spectral quality and reproducible chromatographic performance provided in-depth structural insight into these complex biopharmaceuticals.

Advancements in Native Mass Spectrometry for Antibody Drug Conjugate Characterization

Juan Chavez - Pfizer, Seattle, Washington

Native mass spectrometry (nMS) is an indispensable analytical tool for the molecular characterization of antibody drug conjugates (ADCs), providing valuable information on molecular mass, drug-to-antibody ratio (DAR), and post-translational modifications (PTMs). Native MS of ADCs has primarily utilized quadrupole-time of flight (Q-TOF) mass spectrometers but recent advancements in orbitrap instruments are enabling higher resolution and sensitivity measurements, pushing the boundaries of what is possible. Here we describe the how latest technological advancements in sample handling, fractionation and instrumentation allow nMS to overcome these challenges and support development of novel ADCs

Native MS for AAV Therapeutics: Current Insights and Future Opportunities

Shannon Raab - Eli Lilly and Company, Indianapolis, Indiana

Adeno associated viruses (AAVs) have become widely adopted as delivery vehicles for genomic cargo in gene therapy, but their unique molecular properties present new challenges for therapeutic development. The complexity of AAVs has created an opportunity for advanced mass spectrometry (MS) techniques, particularly native MS combined with charge detection (CD)-MS, to support their characterization. This talk will highlight current findings and discuss future opportunities for leveraging native MS in the characterization of AAV-based therapeutics.

Toward comprehensive characterization of guide RNA and beyond by top-down mass spectrometry

Bifan Chen - Genentech, South San Francisco, California

While mass spectrometry is critical for characterizing oligonucleotide therapeutics, analyzing larger oligonucleotides remains a challenge due to poor spectral quality and ineffective fragmentation. To address this, we developed a rapid online chromatography clean-up method that effectively removes adducts for oligonucleotides up to 200-mers. Furthermore, we implemented a novel low q-CID dissociation strategy that restricts fragmentation to limited cleavages, thereby maximizing sequence coverage. Using a 100-mer sgRNA, this integrated approach demonstrated clean, adduct-free spectra and near-full sequence coverage in a single LC-MS run.

Native Mass Spectrometry for Mapping Antibody Aggregate Species and Comparing Intra- and Extracellular Profiles

Christopher Sauer - Johnson & Johnson Innovative Medicine, Spring House, Pennsylvania

Native mass spectrometry offers a sensitive, non-denaturing platform for identifying and characterizing aggregate species in biotherapeutic antibodies. Here we demonstrate the importance of SEC-MS optimization to enhance detection of intact and subunit-level assemblies while preserving native interactions. Applications include detailed subunit characterization to map aggregate composition and a direct comparison of intra- versus extracellular aggregation profiles, revealing distinct species and likely formation pathways. These findings underscore native MS as a rapid, informative tool for aggregation profiling, root cause of formation and potential for understanding cell machinery.

AiDA Accelerates Top/Middle-Down MS Data Analysis: Application to Deamidation, Isomerization and Oxidation in Biopharmaceuticals

François Griaud - Novartis Pharma AG, Basel, Switzerland

Mass spectrometry (MS) is essential for characterizing biopharmaceuticals, with Top-Down (TD) and Middle-Down (MD) approaches offering information complementary to peptide mapping. Despite achieving high sequence coverage using the combination of fragmentation techniques like HCD, ETD, and UVPD, classical TD/MD data analysis presents several gaps. These include time-consuming fragmentation map creation, difficulty localizing post-translational modifications (PTMs) in non-fragmented regions, overlooked fragmentation pattern differences, and missed diagnostic ions due to monoisotopic peak detection errors in complex TD/MD spectra.
To overcome these issues, the All ion Differential Analysis (AiDA) method was developed for the online characterization of antibody variants, such as methionine oxidation, asparagine deamidation and aspartic acid isomerization, by enabling the rapid identification of diagnostic fragmentation patterns, including the “iso-aspartic acid effect”, at terminal and internal fragments across multiple TD/MD spectra.

Jared Auclair - Northeastern University, Boston, Massachusetts

Jared R. Auclair, PhD is currently the dean of the College of Professional Studies. Auclair has held numerous roles across the university, including Vice Provost of Research Economic Development, Director of Bioinnovation in the Office of the Provost, and Associate Dean of Professional Programs and Graduate Affairs at the College of Science.

As a faculty member in the Department of Chemistry and Chemical Biology, he brings a unique blend of academic expertise and practical innovation to his leadership approach. His research centers on work-integrated credentialed learning and use-inspired research, with a particular emphasis on collaborative work across academic, industry, and government sectors in biopharmaceutical development and analysis.