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. The most direct strategy to evaluate antibody heteromers is via specific enrichment. However, the fact that antibody heterodimers generated from the co-formulated cocktail share highly similar molar mass and size properties as homodimers natively present in each individual antibody drug product poses a unique purification challenge. Here, we report the path to successful enrichment of heterodimers from co-formulated REGEN-COVⓇ and discuss its potential impacts on drug quality.

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 via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

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. Leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis. The utility of this new workflow was demonstrated in three mAb case studies, where different post-translational modifications occurring within the complementarity-determining regions were successfully interrogated for their impact on antigen binding. As this workflow does not require prior enrichment (e.g., by forced degradation or liquid chromatography fractionation) of the variants, it is particularly valuable during the mAb candidate developability assessment, where fast turn-around time is highly desired to assist candidate selection.Abbreviations: ACN: acetonitrile; ADCC: antibody-dependent cell-mediated cytotoxicity; AEX: anion exchange chromatography; bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: critical quality attribute; DDA: data-dependent acquisition; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FA: formic acid; Fab: Fragment antigen-binding; FcRn: neonatal Fc receptor; HC: heavy chain; HIC: hydrophobic interaction chromatography; IAA: iodoacetamide; IEX: ion exchange chromatography; LC: light chain; mAb monoclonal antibody; msAb: monospecific antibody; MS: mass spectrometry; PBS: phosphate-buffered saline; pI: isoelectric point; PTM: post-translational modification; SCX: strong cation exchange chromatography; SEC: size exclusion chromatography; SPR: surface plasmon resonance; XIC: extracted ion chromatography.

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 (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.

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. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

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

Aim: Our objective was to test the feasibility of developing an LC-free, MS-based approach for high-throughput bioanalysis of humanized therapeutic monoclonal antibodies. Methodology: 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. A high-resolution, multiplexed (MSX = 2) parallel reaction monitoring method was developed for data acquisition. Results & conclusion: This proof-of-concept study demonstrated the feasibility of achieving high-throughput MS-based bioanalysis of monoclonal antibodies using an LC-free workflow with sensitivity comparable to conventional LC-MS/MS-based methods.

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. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.

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. Moreover, HILIC-based separation was shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications (PTMs) (e.g. phosphorylation and oxidation) and protein backbone clippings, making it ideally suited for capsid heterogeneity characterization. To overcome the challenges associated with low protein concentrations of AAV samples, as well as the trifluoroacetic acid (TFA)-induced ion suppression during HILIC-MS analysis, different strategies were implemented to improve method sensitivity, including increasing the HILIC column loading and the application of a desolvation gas modification device. Finally, we demonstrated that this integrated HILIC-FLR-MS method can be generically applied to characterize a variety of AAV serotype samples at low concentrations without any sample treatment to achieve unambiguous serotype identification, stoichiometry assessment, and PTM characterization.

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. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity elucidation for the HIC-UV method used in quality control.

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. Here, we report the development of a novel analytical method, which couples mixed-mode size exclusion chromatography (mmSEC) with online native MS detection (mmSEC-MS) for highly sensitive detection and quantitation of homodimer impurities in bsAb samples. Secondary interactions between the protein analytes and the column matrix, which are typically unwanted in SEC applications, are utilized to separate mAb species with similar hydrodynamic volume but different surface characteristics. Using four different bsAbs as testing standards, we demonstrated the versatility of this method in separating homodimer species from bsAb based on either electrostatic interaction or hydrophobic interaction, which was easily achieved by utilizing SEC columns with different properties as well as modulating the salt concentrations. The chromatographic separation between homodimer impurities and bsAb, as achieved by the mmSEC method, was demonstrated to be critical for the improved sensitivity in detecting low-abundance homodimer impurities (LOD from 0.01% to 0.1%). To the best of our knowledge, this newly developed mmSEC-MS method represents the most sensitive MS-based technique in both detection and quantitation of homodimer impurities in bsAb samples.

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. By modifying the desolvation gas with base vapor from triethylamine (TEA), the charge reduction effect can be achieved and utilized to improve the spectral quality from intact mass analysis of protein biopharmaceuticals. The approach and device described in this work suggests a low-cost and practical solution to improve the LC-MS characterization of protein biopharmaceuticals, which has the potential to be widely implemented in biopharmaceutical analytical laboratories.

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. As demonstrated by analyzing mAb molecules with a wide range of pI (isoelectric point) values, the developed method can consistently achieve both high-resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the high sensitivity, a nonconsensus Fab glycosylation site, present at a very low level (<0.1%), was directly detected in the NISTmAb sample without any enrichment. The structure and location of this Fab glycosylation was further characterized by peptide mapping analysis. Despite the extensive characterization of NISTmAb material in previous studies, this is the first time that this Fab-glycosylated variant has been identified in the NISTmAb, demonstrating the value of this new method in achieving a more comprehensive characterization of charge heterogeneity for therapeutic mAbs.

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. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment.

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 "1.2mer"). The interaction between monomer and the extra light chain was characterized by native spray and hydrogen-deuterium exchange mass spectrometry techniques. The goal is to understand the nature of the noncovalent interaction, to map out the interaction interface and regions of potential conformational distortions. In addition, computational modeling was used to aid in binding site identification. The combined results identify the interaction interface to be located in the heavy chain region 38-57 and in the extra light chain region 30-50. To the best of our knowledge, this study is the first to characterize noncovalent interaction of a size variant comprising an antibody monomer and an extra light chain. Structural knowledge generated in this research work is invaluable for process development and construct design of antibody-based biopharmaceuticals.

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). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody-antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.

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. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.

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. Focusing on a class of neurons selective for females, we identified a novel family of vomeronasal ligands, steroid carboxylic acids. These ligands accounted for much of the neuronal activity of urine from some female strains, were necessary for normal levels of male investigatory behavior of female scents, and were sufficient to trigger mounting behavior. CAM represents the first step toward an exhaustive characterization of the molecular cues for natural behavior in a mammalian olfactory system.

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. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

"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 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin.

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin ((10)Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.