Alexey A. Makarov

Conformational stability is a critical attribute for biologics discovery and manufacturability assessment, yet conventional approaches often require substantial material, extensive preparation, or lack sufficient structural resolution for early decision-making. Here, we present a native size-exclusion chromatography hydrogen–deuterium exchange mass spectrometry (SEC-HDX-MS) workflow integrated with molecular modeling to rapidly characterize conformational stability of therapeutic proteins in the discovery space. The workflow was evaluated across monoclonal antibody and bispecific antibody modalities. From a sequence variant comparison, SEC-HDX-MS showed that elevated aggregation did not uniformly reflect global conformational instability; instead, only one variant displayed increased deuterium incorporation, which modeling linked to an elongated, solvent-exposed CDR-H3 loop with increased hydrophobicity. In a representative proprietary mAb (“mAb X”), SEC-HDX-MS revealed that thermally-induced isoaspartate accumulation reduced conformational stability, whereas high-pH stress showed behavior comparable to controls despite higher overall deamidation. Molecular dynamics simulations explained this destabilization through disruption of a backbone Asn43–Ala40 hydrogen bond and altered local coordination after isoAsp formation. Finally, application to a complex bispecific antibody confirmed the workflow’s sensitivity for detecting conformational differences in more complex formats. Collectively, this integrated approach connects global HDX measurements with molecular-level interpretation, requires minimal preparation and limited material, and is compatible with partially purified samples. The workflow enables rapid conformational stability assessment to support candidate ranking, protein engineering, formulation development, and CMC risk assessment during biologics discovery.

Conformational stability is a critical attribute for biologics discovery and manufacturability assessment, yet conventional approaches often require substantial material, extensive preparation, or lack sufficient structural resolution for early decision-making. Here, we present a native size-exclusion chromatography hydrogen–deuterium exchange mass spectrometry (SEC-HDX-MS) workflow integrated with molecular modeling to rapidly characterize conformational stability of therapeutic proteins in the discovery space. The workflow was evaluated across monoclonal antibody and bispecific antibody modalities. From a sequence variant comparison, SEC-HDX-MS showed that elevated aggregation did not uniformly reflect global conformational instability; instead, only one variant displayed increased deuterium incorporation, which modeling linked to an elongated, solvent-exposed CDR-H3 loop with increased hydrophobicity. In a representative proprietary mAb (“mAb X”), SEC-HDX-MS revealed that thermally-induced isoaspartate accumulation reduced conformational stability, whereas high-pH stress showed behavior comparable to controls despite higher overall deamidation. Molecular dynamics simulations explained this destabilization through disruption of a backbone Asn43–Ala40 hydrogen bond and altered local coordination after isoAsp formation. Finally, application to a complex bispecific antibody confirmed the workflow’s sensitivity for detecting conformational differences in more complex formats. Collectively, this integrated approach connects global HDX measurements with molecular-level interpretation, requires minimal preparation and limited material, and is compatible with partially purified samples. The workflow enables rapid conformational stability assessment to support candidate ranking, protein engineering, formulation development, and CMC risk assessment during biologics discovery.

Peptides have become an important modality in the pharmaceutical industry, as they demonstrate heightened potency and target specificity, a property influenced by their conformational structures in solutions. In early drug discovery, structure-based optimization of peptide solution conformation in iterative make-test-design cycles to access peptide drug leads involves large-scale screening. To date, workflows for studying peptide solution conformation reported in the literature are throughput- and resource-limited and do not meet the analytical needs in early discovery space. In this view,context, it is imperative to develop a methodology that exhibits a high analytical throughput, does not involve additional sample preparation steps, and can handle screening of large numbers of samples for solution conformation study. These important analytical requirements in an early discovery pipeline were addressed in this newly developed workflow, where hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insights into the relative solution structural conformation of peptides with ultra-high throughput (uHT) acoustic ejection mass spectrometry (AEMS). The described method exhibits a sampling rate of ∼3 s/sample with very low sample and solvent consumption. The format of this workflow provides the ease and convenience of investigating peptide HDX kinetic profiles, with negligible unwanted back-exchange. The capability of the workflow to discriminate conformational changes was established using myoglobin and apo-myoglobin as protein model systems. Using model peptides, we have further demonstrated the capability of the workflow to differentiate solution structural conformation based on their HDX profiles. This work is an example of a HT HDX-based solution structural conformation workflow that does not require additional sample preparation steps, using uHT AEMS with analytical characteristics suitable for a large sample size screening as part of the down-selection process in the early discovery stage.

High-throughput analysis has become a critical component in chemical biology and analytical chemistry due to the large libraries of compounds that are screened every day for drug development. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein modifications, both chemically deposited and biological post-translational modifications (PTMs). With the advent of antibody drug conjugates (ADCs) and other novel protein-based conjugates, the demand for such an analysis has skyrocketed. Here, we present a new protocol that achieves quantitative data for modified peptides in approximately 30 s of MS acquisition time. This platform includes a direct injection MS approach coupled with new software named iFishMass to extract targeted signals from hundreds of runs. iFishMass automatically generates plots and statistics. This platform will enable a faster analysis of synthetic modifications installed on monoclonal antibodies to create ADCs, and it is potentially scalable to biological PTMs. Sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates, paving the way to an inexpensive but effective platform for high-throughput screening of conjugation sites on proteins.

Proteolytic degradation of recombinant proteins in Chinese hamster ovary (CHO) cells remains a major challenge in biopharmaceutical manufacturing, often reducing product yield and quality. Matriptase‐1, a type II transmembrane serine protease, has been identified as a key contributor to unwanted proteolysis. This study investigates hepatocyte growth factor activator inhibitor‐1 (HAI‐1) overexpression as a strategy to mitigate matriptase‐1‐mediated degradation in CHO cell culture. Using an IL‐12 IgG1 Fc fusion protein and a multi‐specific antibody (M molecule) as model proteins, we employed genetic and biochemical approaches to assess the impact of Chinese hamster HAI‐1 overexpression on protein quality and yield. Our results demonstrate that HAI‐1 overexpression effectively inhibits matriptase‐1 activity, achieving up to a 98% reduction in proteolytic clipping while maintaining cell growth, viability, and product quality. Compared to other protease control strategies, HAI‐1 overexpression presents a practical and scalable solution that does not disrupt essential cellular functions. These findings establish HAI‐1 as a key modulatory tool in CHO‐based protein production, with implications for reducing proteolysis of therapeutic proteins and optimizing biopharmaceutical manufacturing processes.

Biotherapeutics are a prominent class of molecules used to treat a wide range of diseases, including cancer, infections, inflammatory and immune disorders. Advances in recombinant protein engineering have led to the development of therapeutic antibodies with multi-specific attributes and increasingly complex structural and functional formats. The discovery workflow involves high-throughput screening of critical quality attributes using liquid chromatography (LC) to optimize cell line development and facilitate drug candidate selection. However, several LC techniques, such as Size Exclusion Chromatography (SEC) and Hydrophobic Interaction Chromatography (HIC), are not directly compatible with Electrospray Ionization (ESI), hindering mass spectrometry (MS)-based identification of impurity peaks. Consequently, significant time and effort are often required to adapt these method conditions to ESI-MS, which slows down discovery efforts. Matrix-Assisted Laser Desorption Ionization (MALDI) is a surface-based ionization technique that offers an attractive alternative to ESI for high-throughput screening due to its rapid analysis times and resistance to buffer/matrix interferences. MALDI-MS is highly compatible with automated liquid handling tools for sample transfer and requires minimal sample volume. To address the increasingly complex needs and challenging timelines of next-generation biologics development, we have developed an LC-MALDI-MS offline approach for rapid peak identification using an LC-fraction collector and MALDI-TOF-MS. This streamlined approach is adaptable to multiple chromatographic techniques, regardless of their inherent MS compatibility, and is suitable for automation. The workflow is versatile and can be applied universally in combination with various chromatographic techniques and analytes, including monoclonal and bispecific antibodies, fusion proteins, and peptides. Importantly, fraction collection and MS data acquisition can be completed within hours without the need for additional method development or sample preparation for MS-based measurements. Herein, we systematically demonstrate the use of an automated workflow involving SEC, HIC, and Reversed Phase for proteins and peptides, as well as a use case with a bispecific antibody and fusion protein using SEC.