RESUMEN
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (â¼150 kDa) and high structural complexity that require extensive sequence and structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique that sequences and maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications that may occur in bottom-up MS while achieving higher sequence coverage compared to top-down MS. However, returning complete sequence information by MD-MS is still challenging. Here, we show that assigning internal fragments in direct infusion MD-MS of a mAb and an ADC substantially improves their structural characterization. For MD-MS of the reduced NIST mAb, including internal fragments recovers nearly 100% of the sequence by accessing the middle sequence region that is inaccessible by terminal fragments. The identification of important glycosylations can also be improved after the inclusion of internal fragments. For the reduced lysine-linked IgG1-DM1 ADC, we show that considering internal fragments increases the DM1 conjugation sites coverage to 80%, comparable to the reported 83% coverage achieved by peptide mapping on the same ADC (Luo et al. Anal. Chem. 2016, 88, 695-702). This study expands our work on the application of internal fragment assignments in top-down MS of mAbs and ADCs and can be extended to other heterogeneous therapeutic molecules such as multispecifics and fusion proteins for more widespread applications.
Asunto(s)
Anticuerpos Monoclonales , Inmunoconjugados , Anticuerpos Monoclonales/química , Inmunoconjugados/química , Espectrometría de Masas/métodos , Mapeo Peptídico , Lisina/químicaRESUMEN
B3GNT2 is responsible for elongation of cell surface long-chain polylactosamine, which influences the regulation of the immune response, making it an attractive target for immunomodulation. In the development of amide containing B3GNT2 inhibitors guided by structure-based drug design, imidazolones were found to successfully serve as amide bioisosteres. This novel imidazolone isosteric strategy alleviated torsional strain of the amide bond on binding to B3GNT2 and improved potency, isoform selectivity, as well as certain physicochemical and pharmacokinetic properties. Herein, we present the synthesis, SAR, X-ray cocrystal structures, and in vivo PK properties of imidazol-4-ones in the context of B3GNT2 inhibition.
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Amidas , N-Acetilglucosaminiltransferasas , Amidas/farmacología , Amidas/química , N-Acetilglucosaminiltransferasas/metabolismo , Diseño de Fármacos , Relación Estructura-ActividadRESUMEN
Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.
Asunto(s)
Anticuerpos Monoclonales , Descubrimiento de Drogas , Animales , Ratones , Anticuerpos Monoclonales/química , Simulación por Computador , Proteínas Recombinantes , ViscosidadRESUMEN
Membrane proteins and associated complexes currently comprise the majority of therapeutic targets and remain among the most challenging classes of proteins for analytical characterization. Through long-term strategic collaborations forged between industrial and academic research groups, there has been tremendous progress in advancing membrane protein mass spectrometry (MS) analytical methods and their concomitant application to Amgen therapeutic project progression. Herein, I will describe a detailed and personal account of how electrospray ionization (ESI) native mass spectrometry (nMS), ion mobility-MS (IM-MS), reversed phase liquid chromatographic mass spectrometry (RPLC-MS), high-throughput solid phase extraction mass spectrometry, and matrix-assisted laser desorption ionization mass spectrometry methods were developed, optimized, and validated within Amgen Research, and importantly, how these analytical methods were applied for membrane and hydrophobic protein analyses and ultimately therapeutic project support and progression. Additionally, I will discuss all the highly important and productive collaborative efforts, both internal Amgen and external academic, which were key in generating the samples, methods, and associated data described herein. I will also describe some early and previously unpublished nano-ESI (nESI) native-MS data from Amgen Research and the highly productive University of California Los Angeles (UCLA) collaboration. I will also present previously unpublished examples of real-life Amgen biotherapeutic membrane protein projects that were supported by all the MS (and IM) analytical techniques described herein. I will start by describing the initial nESI nMS experiments performed at Amgen in 2011 on empty nanodisc molecules, using a quadrupole time-of-flight MS, and how these experiments progressed on to the 15 Tesla Fourier transform ion cyclotron resonance MS at UCLA. Then described are monomeric and multimeric membrane protein data acquired in both nESI nMS and tandem-MS modes, using multiple methods of ion activation, resulting in dramatic spectral simplification. Also described is how we investigated the far less established and less published subject, that is denaturing RPLC-MS analysis of membrane proteins, and how we developed a highly robust and reproducible RPLC-MS method capable of effective separation of membrane proteins differing in only the presence or absence of an N-terminal post translational modification. Also described is the evolution of the aforementioned RPLC-MS method into a high-throughput solid phase extraction MS method. Finally, I will give my opinion on key developments and how the area of nMS of membrane proteins needs to evolve to a state where it can be applied within the biopharmaceutical research environment for routine therapeutic project support.
Asunto(s)
Proteínas de la Membrana , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en TándemRESUMEN
Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y - 1/x ions, while isoAsp-containing peptides yielded more enhanced y - 18/y - 45/y - 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.
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Ácido Isoaspártico , Péptidos , Ácido Isoaspártico/análisis , Ácido Isoaspártico/química , Secuencia de Aminoácidos , Espectrometría de Masas/métodos , Péptidos/química , Ácido Aspártico/química , Iones , Rayos UltravioletaRESUMEN
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.
Asunto(s)
Espectrometría de Masas , Anticuerpos Monoclonales/química , Humanos , Glicosilación , Espectrometría de Masas/métodos , Disulfuros/química , Lisina/químicaRESUMEN
Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values.
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Cromatografía de Fase Inversa , Extracción en Fase Sólida , Espectrometría de Masas/métodos , Cromatografía de Fase Inversa/métodos , Peso Molecular , Anticuerpos Monoclonales/químicaRESUMEN
Isomeric peptide analyses are an analytical challenge of great importance to therapeutic monoclonal antibody and other biotherapeutic product development workflows. Aspartic acid (Asp, D) to isoaspartic acid (isoAsp, isoD) isomerization is a critical quality attribute (CQA) that requires careful control, monitoring, and quantitation during the drug discovery and production processes. While the formation of isoAsp has been implicated in a variety of disease states such as autoimmune diseases and several types of cancer, it is also understood that the formation of isoAsp results in a structural change impacting efficacy, potency, and immunogenic properties, all of which are undesirable. Currently, lengthy ultrahigh-performance liquid chromatography (UPLC) separations are coupled with MS for CQA analyses; however, these measurements often take over an hour and drastically limit analysis throughput. In this manuscript, drift tube ion mobility spectrometry-mass spectrometry (DTIMS-MS) and both a standard and high-resolution demultiplexing approach were utilized to study eight isomeric Asp and isoAsp peptide pairs. While the limited resolving power associated with the standard DTIMS analysis only separated three of the eight pairs, the application of HRdm distinguished seven of the eight and was only unable to separate DL and isoDL. The rapid high-throughput HRdm DTIMS-MS method was also interfaced with both flow injection and an automated solid phase extraction system to present the first application of HRdm for isoAsp and Asp assessment and demonstrate screening capabilities for isomeric peptides in complex samples, resulting in a workflow highly suitable for biopharmaceutical research needs.
Asunto(s)
Espectrometría de Movilidad Iónica , Ácido Isoaspártico , Cromatografía Liquida , Espectrometría de Movilidad Iónica/métodos , Ácido Isoaspártico/análisis , Espectrometría de Masas/métodos , PéptidosRESUMEN
Mass spectrometry (MS) plays a key role throughout all stages of drug development and is now as ubiquitous as other analytical techniques such as surface plasmon resonance, nuclear magnetic resonance, and supercritical fluid chromatography, among others. Herein, we aim to discuss the history of MS, both electrospray and matrix-assisted laser desorption ionization, specifically for the analysis of antibodies, evolving through to denaturing and native-MS analysis of newer biologic moieties such as antibody-drug conjugates, multispecific antibodies, and interfering nucleic acid-based therapies. We discuss challenging therapeutic target characterization such as membrane protein receptors. Importantly, we compare and contrast the MS and hyphenated analytical chromatographic methods used to characterize these therapeutic modalities and targets within biopharmaceutical research and highlight the importance of appropriate MS deconvolution software and its essential contribution to project progression. Finally, we describe emerging applications and MS technologies that are still predominantly within either a development or academic stage of use but are poised to have significant impact on future drug development within the biopharmaceutic industry once matured. The views reflected herein are personal and are not meant to be an exhaustive list of all relevant MS performed within biopharmaceutical research but are what we feel have been historically, are currently, and will be in the future the most impactful for the drug development process.
Asunto(s)
Descubrimiento de Drogas/métodos , Proteínas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anticuerpos Monoclonales/análisis , Automatización de Laboratorios , Biofarmacia/métodos , Cromatografía Liquida , Industria Farmacéutica/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Inmunoconjugados/análisis , Inmunoconjugados/química , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray/historia , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/historia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentaciónRESUMEN
Aspartic acid (Asp) to isoaspartic acid (isoAsp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of isoAsp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the isoAsp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the isoAsp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the isoAsp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented isoAsp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the isoAsp containing peptide, separated by rpHPLC, results in LC-MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.
Asunto(s)
Ácido Aspártico/análisis , Ácido Aspártico/química , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Ácido Isoaspártico/análisis , Ácido Isoaspártico/química , Péptidos/análisis , Péptidos/normas , Espectrometría de Masas en TándemRESUMEN
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media-quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle-based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non-denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single-stranded oligonucleotide in high purity (92%) under denaturing conditions. The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía por Intercambio Iónico , G-Cuádruplex , Espectrometría de Masas , Oligonucleótidos/análisisRESUMEN
Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.
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Ciclotrones , Análisis de Fourier , Inmunoconjugados/química , Espectrometría de Masas/métodos , Sales (Química)/química , Anticuerpos Monoclonales/química , Cesio/química , Chaperonina 60/química , Inmunoglobulina G/química , Cadenas kappa de Inmunoglobulina/química , Yoduros/química , Maitansina/química , Peso Molecular , ARN Interferente Pequeño/químicaRESUMEN
KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.
RESUMEN
Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH]n+ or negative [M - nH]n- depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; -203.1 Da).
Asunto(s)
Algoritmos , Biofarmacia/métodos , Peso Molecular , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Anticuerpos Monoclonales/química , Glicosilación , Inmunoconjugados/química , Lisina/química , Proteínas de la Membrana/químicaRESUMEN
One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
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Benchmarking , Espectrometría de Masas/métodos , Proteínas/química , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
The potential repertoire of short interfering RNA (siRNA) therapeutics is expanding as targeting strategies evolve. One approach to enable organ-specific delivery has been to directly conjugate siRNA to a monoclonal antibody (siRNA-mAb), analogous to antibody-drug conjugates. Detection of intact siRNA-mAb conjugates presents a bioanalytical challenge given that certain synthetic nucleotide chemical modifications and low-temperature requirements render common oligonucleotide detection assays, such as reverse transcription-polymerase chain reaction, incompatible with the immunoassay component. To circumvent these issues, we developed a triplex-forming oligonucleotide ELISA using locked nucleic acid (LNA) containing oligonucleotide probes. We demonstrate that the incorporation of these LNAs allow for an enrichment and immobilization of siRNA directly conjugated to an antibody at nondenaturing temperatures. Without further requirement for extraction or amplification, we can sensitively and specifically detect intact siRNA-mAb conjugates in complex matrices such as serum and tissue homogenate.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Oligonucleótidos/aislamiento & purificación , ARN Interferente Pequeño/aislamiento & purificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Oligonucleótidos/genética , Oligonucleótidos/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunologíaRESUMEN
Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.
RESUMEN
Antibody-drug conjugates (ADCs) are antibody-based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine-targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)-MS and gas-phase unfolding for the structural characterization of lysine-linked monoclonal antibody (mAb)-biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography-Mass Spectrometry (LC-MS) measurements, we performed both size exclusion chromatography (SEC) and native IM-MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross-sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody-biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb-biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM-MS and CIU technologies on the future of ADC development pipelines.