Benchmarking glycoform-resolved affinity separation – mass spectrometry assays for studying FcγRIIIa binding
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ABSTRACT:
SUBMITTER: Gstottner C
PROVIDER: S-EPMC10925690 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Determination of the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure-function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.
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Project description:The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the FcγIIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala - Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships.
| S-EPMC9494200 | biostudies-literature
Project description:Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).
| S-EPMC8741291 | biostudies-literature
Project description:The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb contributes to neutrophil activation and is involved in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These processes present important mechanisms-of-actions of therapeutic antibodies. The very low affinity of IgG toward FcɣRIIIb (KD ~ 10 µM) is a technical challenge for interaction studies. Additionally, the interaction is strongly dependent on IgG glycosylation, a major contributor to proteoform heterogeneity. We developed an affinity chromatography-mass spectrometry (AC-MS) assay for analyzing IgG-FcɣRIIIb interactions in a proteoform-resolved manner. This proved to be well suited to study low-affinity interactions. The applicability and selectivity of the method were demonstrated on a panel of nine different IgG monoclonal antibodies (mAbs), including no-affinity, low-affinity and high-affinity Fc-engineered or glycoengineered mAbs. Thereby, we could reproduce reported affinity rankings of different IgG glycosylation features and IgG subclasses. Additional post-translational modifications (IgG1 Met252 oxidation, IgG3 hinge-region O-glycosylation) showed no effect on FcɣRIIIb binding. Interestingly, we observed indications of an effect of the variable domain sequence on the Fc-binding that deserves further attention. Our new AC-MS method is a powerful tool for expanding knowledge on structure-function relationships of the IgG-FcɣRIIIb interaction. Hence, this assay may substantially improve the efficiency of assessing critical quality attributes of therapeutic mAbs with respect to an important aspect of neutrophil activation.
| S-EPMC8726612 | biostudies-literature
Project description:High-throughput identification and quantification of protein/peptide biomarkers from biofluids in a label-free manner is achieved by interfacing bio-affinity arrays (BAAs) with nano-electrospray desorption electrospray ionization mass spectrometry (nano-DESI-MS). A wide spectrum of proteins and peptides ranging from phosphopeptides to cis-diol biomolecules as well as thrombin can be rapidly extracted via arbitrarily predefined affinity interactions including coordination chemistry, covalent bonding, and biological recognition. An integrated MS platform allows continuous interrogation. Profiling and quantitation of dysregulated phosphopeptides from small-volume (∼5 μL) serum samples has been successfully demonstrated. As a front-end device adapted to any mass spectrometer, this MS platform might hold much promise in protein/peptide analysis in point-of-care (POC) diagnostics and clinical applications.
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Project description:The glycoform of a therapeutic monoclonal antibody (mAb) has a significant impact on its effector function as well as its safety and pharmacokinetics. Glycoform heterogeneity is influenced by various factors, including the producing cells and cell culture processes. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) and applied the technique to the glycan structural analysis of mAbs. To achieve both the highest sensitivity and the most comprehensive glycan profiling, we integrated our energy-resolved oxonium ion monitoring (Erexim) method with SFC-MS to construct a new SFC-Erexim technology. An 8 min analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-Erexim detected a total of 102 glycoforms, with a detection limit of 5 attomoles. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-Erexim compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. The SFC-MS-based glycoform profiling method will provide an ideal platform for the in-depth analysis of precise glycan structure and abundance.
| S-EPMC9685587 | biostudies-literature
Project description:Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.
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Project description:The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and-only if a sublibrary is active-additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.
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Project description:The biological functions of lipids are entirely dependent on their molecular structures with even small changes in structure─such as different sites of unsaturation─providing critical markers for changes in the underlying metabolism. Conventional mass spectrometry imaging (MSI) approaches, however, face the twin challenges of mixture and structural complexity and are typically unable to differentiate lipid isomers that differ only in the position(s) of carbon-carbon double bonds. Recent coupling of ozone-induced dissociation (OzID) with matrix-assisted laser desorption/ionization (MALDI)-MSI has demonstrated the potential to map changes in individual double-bond isomers, thus enabling visualization of the modulation in lipid desaturation in adjacent tissue types. This has, to date, only been performed in positive-ion mode due to a generally higher abundance of phosphatidylcholines (PC) in mammalian tissues and the efficient desorption/ionization of this lipid subclass. Many other glycerophospholipids (GPLs), however, are better detected in negative-ion mode as deprotonated anions. Recently, OzID has been implemented on a traveling-wave ion-mobility mass spectrometer (Waters, SYNAPT G2-Si) that provides a 50-fold increase in the rate of the gas-phase reaction between ionized lipids and ozone and a commensurate increase in sensitivity for isomer-resolved mass spectrometry. These gains are exploited here to interrogate the distributions of anionic GPL isomers in biological tissues, covering the subclasses phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). Exploiting both ozone- and collision-induced dissociation in a single acquisition simultaneously identifies sites of unsaturation and acyl chain composition from the same mass spectrum.
| S-EPMC10557375 | biostudies-literature
Project description:Fcɤ receptors (FcɤR) mediate key functions in immunological responses. For instance, FcɤRIIIa is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). FcɤRIIIa interacts with the fragment crystallizable (Fc) of immunoglobulin G (IgG). This interaction is known to be highly dependent on IgG Fc glycosylation. Thus, the impact of glycosylation features on this interaction has been investigated in several studies by numerous analytical and biochemical techniques. FcɤRIIIa affinity chromatography (AC) hyphenated to mass spectrometry (MS) is a powerful tool to address co-occurring Fc glycosylation heterogeneity of monoclonal antibodies (mAbs). However, MS analysis of mAbs at the intact level may provide limited proteoform resolution, for example, when additional heterogeneity is present, such as antigen-binding fragment (Fab) glycosylation. Therefore, we investigated middle-up approaches to remove the Fab and performed AC-MS on the IgG Fc to evaluate its utility for FcɤRIIIa affinity assessment compared to intact IgG analysis. We found the protease Kgp to be particularly suitable for a middle-up FcɤRIIIa AC-MS workflow as demonstrated for the Fab glycosylated cetuximab. The complexity of the mass spectra of Kgp digested cetuximab was significantly reduced compared to the intact level while affinity was fully retained. This enabled a reliable assignment and relative quantitation of Fc glycoforms in FcɤRIIIa AC-MS. In conclusion, our workflow allows a functional separation of differentially glycosylated IgG Fc. Consequently, applicability of FcɤRIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.
| S-EPMC6822288 | biostudies-literature