RESUMEN
Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.
Asunto(s)
Electroforesis Capilar/instrumentación , Dispositivos Laboratorio en un Chip , Espectrometría de Masas/instrumentación , Polisacáridos/análisis , Proteínas Recombinantes de Fusión , Glicosilación , Mapeo Peptídico/instrumentación , Mapeo Peptídico/métodos , Polisacáridos/química , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.
Asunto(s)
Medición de Intercambio de Deuterio , Mapeo Epitopo/métodos , Epítopos/análisis , Nivolumab/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/química , Sitios de Unión , Cromatografía Líquida de Alta Presión , Epítopos/química , Epítopos/inmunología , Humanos , Simulación del Acoplamiento Molecular , Nivolumab/metabolismo , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Espectrometría de Masas en TándemRESUMEN
We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Hidrógeno/química , Interleucina-7/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Receptores de Interleucina-7/metabolismo , Humanos , Cinética , Unión Proteica , Conformación ProteicaRESUMEN
Higher-order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 â¼ 6.2 pM vs Kd, Adnectin2 â¼ 46 nM). Furthermore, the study evaluates the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adnectin1 and adnectin2 binding, is a flexible loop that connects two ß-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with apparently similar biophysical properties.
Asunto(s)
Mapeo Epitopo , Huella de Proteína , Receptores de Interleucina-6/química , Medición de Intercambio de Deuterio , Humanos , Espectrometría de Masas , Unión Proteica , Conformación ProteicaRESUMEN
Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.
Asunto(s)
Neoplasias , Microambiente Tumoral , Animales , Concentración de Iones de Hidrógeno , Ipilimumab/uso terapéutico , Ratones , Índice TerapéuticoRESUMEN
Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.25:1 to 4:1 to make two sets of samples. The resulting mixtures were analyzed by microcapillary LC-FTMS and dMS. dMS successfully identified five out of the six peptides as statistically significant differences (p Asunto(s)
Mezclas Complejas/química
, Péptidos/química
, Proteínas/química
, Espectroscopía Infrarroja por Transformada de Fourier
, Secuencia de Aminoácidos
, Animales
, Bovinos
, Cromatografía Líquida de Alta Presión
, Humanos
, Datos de Secuencia Molecular
, Mapeo Peptídico
, Ratas
, Reproducibilidad de los Resultados
RESUMEN
BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.
Asunto(s)
Anticuerpos Monoclonales/química , Asparagina/análisis , Ácido Aspártico/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/sangre , Cromatografía Liquida/métodos , Humanos , Inmunoprecipitación/métodos , Isomerismo , Macaca fascicularis , Masculino , Espectrometría de Masas en Tándem/métodosRESUMEN
Antibody-drug conjugates (ADCs) are emerging modalities in the pharmaceutical industry. Characterization of ADC's drug-to-antibody ratio (DAR) becomes a key assessment because of its importance in ADC efficacy and safety. DAR characterization by conventional intact protein MS analysis, however, is challenging because of high heterogeneity of ADC samples. The analysis often requires protein deglycosylation, disulfide-bond reduction, or partial fragmentation. In this study, we illustrate the practical utility of ion mobility mass spectrometry (IM-MS) in a routine LC/MS workflow for DAR measurements. This strategy allows analyte "cleanup" in the gas phase, providing significant improvement of signal-to-noise ratios of ADC intact mass spectra for accurate DAR measurements. In addition, protein drift time analysis offers a new dimension in monitoring the changes of DAR in lot-to-lot analysis.
Asunto(s)
Inmunoconjugados/análisis , Inmunoconjugados/química , Espectrometría de Masas/métodos , Cromatografía Líquida de Alta Presión , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/químicaRESUMEN
Disease modifying treatments for Alzheimer's disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Proteína C-Reactiva/líquido cefalorraquídeo , Espectrometría de Masas , Factores de Crecimiento Nervioso/líquido cefalorraquídeo , Proteínas del Tejido Nervioso/líquido cefalorraquídeo , Proteómica , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Estradiol/metabolismo , Espectrometría de Masas/métodos , Fenoles/metabolismo , Plasma/química , Pirazoles/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Femenino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Datos de Secuencia Molecular , Tamaño de los Órganos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/metabolismo , Útero/anatomía & histologíaRESUMEN
Efficiently identifying and quantifying disease- or treatment-related changes in the abundance of proteins is an important area of research for the pharmaceutical industry. Here we describe an automated, label-free method for finding differences in complex mixtures using complete LC-MS data sets, rather than subsets of extracted peaks or features. The method selectively finds statistically significant differences in the intensity of both high-abundance and low-abundance ions, accounting for the variability of measured intensities and the fact that true differences will persist in time. The method was used to compare two complex peptide mixtures with known peptide differences. This controlled experiment allowed us to assess the validity of each difference found and so to analyze the method's sensitivity and specificity. The method detects both presence versus absence and a 2-fold change in peptide concentration near the limit of detection of the instrument used, where chromatographic peaks may not be sufficiently well defined to be detected in individual samples. The method is more sensitive and gives fewer false positives than subtractive methods that ignore signal variability. Differential mass spectrometry combined with targeted MS/MS analysis of only identified differences may save both computation time and human effort compared to shotgun proteomics approaches.