A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies.

The clinical use of interleukin-2 (IL-2) for cancer immunotherapy is limited by severe toxicity. Emerging IL-2 therapies with reduced IL-2 receptor alpha (IL-2Rα) binding aim to mitigate toxicity and regulatory T cell (Treg) expansion but have had limited clinical success. Here, we show that IL-2Rα engagement is critical for the anti-tumor activity of systemic IL-2 therapy.

Distinct chemical degradation pathways of AAV1 and AAV8 under thermal stress conditions revealed by analytical anion exchange chromatography and LC-MS-based peptide mapping.

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity.

Enrichment strategy and initial characterization of heterodimers enriched from a co-formulated cocktail of therapeutic antibodies against SARS-COV-2.

Co-formulation of multiple drug products is an efficient and convenient approach to simultaneously deliver multiple biotherapeutics with the potentially added benefit of a synergistic therapeutic effect. However, co-formulation also increases the risk of heteromeric interactions, giving rise to unique impurities with unknown efficacy and immunogenicity. Therefore, it is critical to develop methods to evaluate the risk of heteromers as an impurity that could affect potency, efficacy, and/or immunogenicity.

Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed an imine bond between lysine and serine residues.

A competitive binding-mass spectrometry strategy for high-throughput evaluation of potential critical quality attributes of therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) have a propensity to host a large number of chemical and enzymatical modifications that need to be properly assessed for their potential impact on target binding. Traditional strategies of assessing the criticality of these attributes often involve a laborious and low-throughput variant enrichment step prior to binding affinity measurement. Here, we developed a novel competitive binding-based enrichment strategy followed by mass spectrometry analysis (namely, competitive binding-MS) to achieve high-throughput evaluation of potential critical quality attributes in therapeutic mAbs.

Coupling Anion Exchange Chromatography with Native Mass Spectrometry for Charge Heterogeneity Characterization of Monoclonal Antibodies.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (ps), it is an attractive alternative for charge variant analysis of IgG4 molecules.

Post-Column Denaturation-Assisted Native Size-Exclusion Chromatography-Mass Spectrometry for Rapid and In-Depth Characterization of High Molecular Weight Variants in Therapeutic Monoclonal Antibodies.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples.

Development of a chromatography-free method for high-throughput MS-based bioanalysis of therapeutic monoclonal antibodies.

Our objective was to test the feasibility of developing an LC-free, MS-based approach for high-throughput bioanalysis of humanized therapeutic monoclonal antibodies. A universal tryptic peptide from human IgG1, IgG3 and IgG4 was selected as the surrogate peptide for quantitation. After tryptic digestion, the surrogate peptide was fractionated via solid-phase extraction before being subjected to direct infusion-based MS/MS analysis.

Versatile, Sensitive, and Robust Native LC-MS Platform for Intact Mass Analysis of Protein Drugs.

Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications.

Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry.

To support adeno-associated virus (AAV)-based gene therapy development, characterization of the three capsid viral proteins (VP; VP1/VP2/VP3) from recombinant AAV can offer insights on capsid identity, heterogeneity, and product and process consistency. Intact protein mass analysis is a rapid, reliable, and sensitive method to confirm AAV serotypes based on accurate mass measurement of the constituent capsid proteins. Compared to commonly applied reversed-phase liquid chromatography (RPLC) methods, we demonstrated that, using a wide-pore amide-bonded column, hydrophilic interaction chromatography (HILIC) could achieve improved separation of VPs from a variety of AAV serotypes using a generic method prior to MS detection.

Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products.

Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization.

Coupling Mixed-Mode Size Exclusion Chromatography with Native Mass Spectrometry for Sensitive Detection and Quantitation of Homodimer Impurities in Bispecific IgG.

Detection and quantitation of homodimer impurities in therapeutic bispecific antibody (bsAb) drug products is essential to support development and quality control (QC) release. LC-MS-based techniques have been frequently applied for this analysis. However, sensitive detection of low-abundance homodimer impurities can still be challenging for regular workflows, which is largely due to the lack of chromatographic resolution between the impurities and the main bsAb species.

Simple Approach for Improved LC-MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas.

LC-MS based analysis of protein biopharmaceuticals could benefit from improved data quality, which can subsequently lead to improved drug characterization with higher confidence and less ambiguity. In this study, we created a simple device to modify the desolvation gas on a Q-Exactive mass spectrometer and to demonstrate the utility in improving both peptide mapping analysis and intact mass analysis, the two most routinely and widely applied LC-MS techniques in protein biopharmaceutical characterization. By modifying the desolvation gas with acid vapor from propionic acid (PA) and isopropanol (IPA), the ion suppression effects from trifluoroacetic acid (TFA) in a typical peptide mapping method can be effectively mitigated, thus leading to improved MS sensitivity.

Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry.

In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb molecule is often associated with critical quality attributes and is therefore monitored throughout development and during QC release to ensure product and process consistency. Elucidating the cause of each charge variant species is an involved process that often requires offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS) analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In this study, we have developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis (SCX-MS) optimized for mAb separation and detection.

Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry.

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample.

Mapping the Binding Interface in a Noncovalent Size Variant of a Monoclonal Antibody Using Native Mass Spectrometry, Hydrogen-Deuterium Exchange Mass Spectrometry, and Computational Analysis.

Variants of monoclonal antibody containing an extra light chain have been reported in protein products. Due to potential impact on potency and immunogenicity, it is important to understand the formation mechanism of such variants so that appropriate control strategies can be implemented to assure product quality. In a model monoclonal antibody, we observed a size variant with an extra light chain noncovalently associated with the monomer (later named as "1.

Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification-Structure-Function Correlations by Hydrogen-Deuterium Exchange Mass Spectrometry.

Chemical modifications can potentially change monoclonal antibody's (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen-deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1).

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level.

A Molecular Code for Identity in the Vomeronasal System.

In social interactions among mammals, individuals are recognized by olfactory cues, but identifying the key signals among thousands of compounds remains a major challenge. To address this need, we developed a new technique, component-activity matching (CAM), to select candidate ligands that "explain" patterns of bioactivity across diverse complex mixtures. Using mouse urine from eight different sexes and strains, we identified 23 components to explain firing rates in seven of eight functional classes of vomeronasal sensory neurons.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter.

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.