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[This corrects the article DOI: 10.1371/journal.pbio.2002457.].
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A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.
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Lipidómica , Espectrometría de Masas en Tándem , Animales , Cromatografía Liquida , Lípidos , Ratones , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
INTRODUCTION: Biomarkers that reflect pathologic processes affecting neuronal function during preclinical and early stages of Alzheimer's disease (AD) are needed to aid drug development. METHODS: A targeted, stable isotope, quantitative mass spectrometry-based investigation of longitudinal changes in concentrations of previously identified candidate biomarkers was performed in cerebrospinal fluid (CSF) of Alzheimer's Disease Neuroimaging Initiative participants who were classified as cognitively normal (CN; n = 76) or with mild cognitive impairment (MCI; n = 111) at baseline. RESULTS: Of the candidate biomarkers, the CSF concentration of neuronal pentraxin 2 (NPTX2), a protein involved in synaptic function, exhibited rates of change that were significantly different between three comparison groups (i.e., CN vs. MCI participants; AD pathology positive vs. negative defined by phosphorylated tau181/amyloid beta1-42 ratio; and clinical progressors vs. non-progressors). The rate of change of NPTX2 also significantly correlated with declining cognition. DISCUSSION: CSF NPTX2 concentration is a strong prognostic biomarker candidate of accelerated cognitive decline with potential use as a therapeutic target.
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Enfermedad de Alzheimer , Biomarcadores/líquido cefalorraquídeo , Proteína C-Reactiva/líquido cefalorraquídeo , Disfunción Cognitiva , Proteínas del Tejido Nervioso/líquido cefalorraquídeo , Proteómica , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/líquido cefalorraquídeo , Disfunción Cognitiva/líquido cefalorraquídeo , Disfunción Cognitiva/patología , Humanos , Estudios Longitudinales , Espectrometría de Masas , Fosforilación , Proteínas tau/líquido cefalorraquídeoRESUMEN
Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.
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Dolor/metabolismo , Receptor EphB2/metabolismo , Receptores de N-Metil-D-Aspartato/análisis , Animales , Células HEK293 , Humanos , Ratones , Neuronas/metabolismo , Fosforilación , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Análisis de Secuencia de Proteína , Médula Espinal/metabolismo , Médula Espinal/patología , Tirosina/metabolismoRESUMEN
Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng µL-1 compared to 0.05 ng µL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.
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Cromatografía de Afinidad/métodos , Enzimas Inmovilizadas/química , Membranas Artificiales , Trastuzumab/análisis , Secuencia de Aminoácidos , Animales , Masculino , Prueba de Estudio Conceptual , Proteolisis , Ratas Sprague-Dawley , Proteína Estafilocócica A/química , Factores de Tiempo , Trastuzumab/química , Trastuzumab/aislamiento & purificación , Trastuzumab/farmacocinética , Tripsina/químicaRESUMEN
Advances in mass spectrometry, proteomics, protein bioanalytical approaches, and biochemistry have led to a rapid evolution and expansion in the area of mass spectrometry-based biomarker discovery and development. The last decade has also seen significant progress in establishing accepted definitions, guidelines, and criteria for the analytical validation, acceptance and qualification of biomarkers. These advances have coincided with a decreased return on investment for pharmaceutical research and development and an increasing need for better early decision making tools. Empowering development teams with tools to measure a therapeutic interventions impact on disease state and progression, measure target engagement and to confirm predicted pharmacodynamic effects is critical to efficient data-driven decision making. Appropriate implementation of a biomarker or a combination of biomarkers can enhance understanding of a drugs mechanism, facilitate effective translation from the preclinical to clinical space, enable early proof of concept and dose selection, and increases the efficiency of drug development. Here we will provide descriptions of the different classes of biomarkers that have utility in the drug development process as well as review specific, protein-centric, mass spectrometry-based approaches for the discovery of biomarkers and development of targeted assays to measure these markers in a selective and analytically precise manner.
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Biomarcadores , Desarrollo de Medicamentos , Espectrometría de Masas , ProteómicaRESUMEN
Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.
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Anticuerpos Monoclonales/sangre , Bioensayo/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/química , Macaca mulatta , Proteolisis , Tripsina/químicaRESUMEN
Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.
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Anticuerpos Monoclonales/farmacocinética , Análisis Químico de la Sangre/instrumentación , Péptidos/análisis , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/sangre , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Semivida , Inmunoglobulina G/metabolismo , Inmunoprecipitación , Marcaje Isotópico , Macaca fascicularis , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
BACKGROUND: Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. METHODS: IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. RESULTS: The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R(2) = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. CONCLUSIONS: Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.
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Proteínas tau/líquido cefalorraquídeo , Anticuerpos , Estudios de Casos y Controles , Cromatografía Liquida/métodos , Humanos , Isoformas de Proteínas/líquido cefalorraquídeo , Isoformas de Proteínas/inmunología , Espectrometría de Masas en Tándem/métodos , Proteínas tau/inmunologíaRESUMEN
The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched (13)C6(15)N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically "heavy" proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2',3'-cyclic nucleotide 3'-phosphodiesterase.
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Núcleo Celular/metabolismo , Proteómica , Sueño/fisiología , Área Tegmental Ventral/citología , Aminoácidos/metabolismo , Animales , Animales Recién Nacidos , Peso Corporal/efectos de los fármacos , Femenino , Péptidos y Proteínas de Señalización Intracelular/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Tamaño de la Camada/efectos de los fármacos , Lisina/administración & dosificación , Masculino , Neuropéptidos/administración & dosificación , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/química , Orexinas , Péptidos/administración & dosificación , Embarazo , Mapas de Interacción de Proteínas/genética , Proteínas/metabolismo , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Advances in mass spectrometry, proteomics, protein bioanalytical approaches, and biochemistry have led to a rapid evolution and expansion in the area of mass spectrometry-based biomarker discovery and development. The last decade has also seen significant progress in establishing accepted definitions, guidelines, and criteria for the analytical validation, acceptance, and qualification of biomarkers. These advances have coincided with a decreased return on investment for pharmaceutical research and development and an increasing need for better early decision making tools. Empowering development teams with tools to measure a therapeutic interventions impact on disease state and progression, measure target engagement, and to confirm predicted pharmacodynamic effects is critical to efficient data-driven decision making. Appropriate implementation of a biomarker or a combination of biomarkers can enhance understanding of a drugs mechanism, facilitate effective translation from the preclinical to clinical space, enable early proof of concept and dose selection, and increase the efficiency of drug development. Here we will provide descriptions of the different classes of biomarkers that have utility in the drug development process as well as review specific, protein-centric, mass spectrometry-based approaches for the discovery of biomarkers and development of targeted assays to measure these markers in a selective and analytically precise manner.
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Biomarcadores/metabolismo , Descubrimiento de Drogas/métodos , Espectrometría de Masas/métodos , Farmacocinética , Animales , HumanosRESUMEN
Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.
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Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL(3). The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 --> 3)-Gal-beta(1 --> 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.
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Apolipoproteína C-III/aislamiento & purificación , HDL-Colesterol/química , Espectrometría de Masas/métodos , Proteómica/métodos , Secuencia de Aminoácidos , Apolipoproteína C-III/análisis , Apolipoproteína C-III/genética , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificaciónRESUMEN
Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an â¼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.
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Human respiratory syncytial virus (RSV) causes a substantial proportion of respiratory tract infections worldwide. Although RSV reinfections occur throughout life, older adults, particularly those with underlying comorbidities, are at risk for severe complications from RSV. There is no RSV vaccine available to date, and treatment of RSV in adults is largely supportive. A correlate of protection for RSV has not yet been established, but antibodies targeting the pre-fusion conformation of the RSV F glycoprotein play an important role in RSV neutralization. We previously reported a Phase 1 study of an mRNA-based vaccine (V171) expressing a pre-fusion-stabilized RSV F protein (mDS-Cav1) in healthy adults. Here, we evaluated an mRNA-based vaccine (V172) expressing a further stabilized RSV pre-fusion F protein (mVRC1). mVRC1 is a single chain version of RSV F with interprotomer disulfides in addition to the stabilizing mutations present in the mDS-Cav1 antigen. The immunogenicity of the two mRNA-based vaccines encoding mVRC1 (V172) or a sequence-optimized version of mDS-Cav1 to improve transcriptional fidelity (V171.2) were compared in RSV-naïve and RSV-experienced African green monkeys (AGMs). V172 induced higher neutralizing antibody titers than V171.2 and demonstrated protection in the AGM challenge model. We conducted a Phase 1, randomized, placebo-controlled, clinical trial of 25 µg, 100 µg, 200 µg, or 300 µg of V172 in healthy older adults (60-79 years old; N = 112) and 100 µg, 200 µg, or 300 µg of V172 in healthy younger adults (18-49 years old; N = 48). The primary clinical objectives were to evaluate the safety and tolerability of V172, and the secondary objective was to evaluate RSV serum neutralization titers. The most commonly reported solicited adverse events were injection-site pain, injection-site swelling, headache, and tiredness. V172 was generally well tolerated in older and younger adults and increased serum neutralizing antibody titers, pre-fusion F-specific competing antibody titers, and RSV F-specific T-cell responses.
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The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.
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Cromatografía , Vacunas , Biomarcadores , Tratamiento Basado en Trasplante de Células y Tejidos , Espectrometría de Masas , Oligonucleótidos , TecnologíaRESUMEN
Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral ß amyloid (Aß) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.
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The quantitation of therapeutic antibodies by mass spectrometry often utilizes a surrogate peptide approach following enzymatic digestion of the antibody. Although this approach has been widely adopted, it is labor intensive with limited throughput in most instances. In addition, this approach can pose challenges when attempting to infer details such as quantity and modification state of the intact analyte. Recent enhancements in instrumentation and sample preparation have enabled quantitation through mass spectrometry detection of the intact protein circumnavigating many limitations of the surrogate peptide approach. Presented here is a method for quantitative analysis of therapeutic monoclonal antibodies (mAb) at the fully intact level in a complex pharmacokinetic study. This methodology yielded sensitivity down to 0.1µg/mL from 30µL of a biological sample volume to be utilized across multiple preclinical species without the need for pooling.
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Espectrometría de Masas , Anticuerpos Monoclonales , PéptidosRESUMEN
Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.
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Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Anciano , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Humanos , Inmunogenicidad Vacunal , Persona de Mediana Edad , ARN Mensajero , Proteínas Virales de FusiónRESUMEN
BACKGROUND: Neutralizing mAbs can prevent communicable viral diseases. MK-1654 is a respiratory syncytial virus (RSV) F glycoprotein neutralizing monoclonal antibody (mAb) under development to prevent RSV infection in infants. Development and validation of methods to predict efficacious doses of neutralizing antibodies across patient populations exposed to a time-varying force of infection (i.e., seasonal variation) are necessary. METHODS: Five decades of clinical trial literature were leveraged to build a model-based meta-analysis (MBMA) describing the relationship between RSV serum neutralizing activity (SNA) and clinical endpoints. The MBMA was validated by backward translation to animal challenge experiments and forward translation to predict results of a recent RSV mAb trial. MBMA predictions were evaluated against a human trial of 70 participants who received either placebo or one of four dose-levels of MK-1654 and were challenged with RSV [NCT04086472]. The MBMA was used to perform clinical trial simulations and predict efficacy of MK-1654 in the infant target population. FINDINGS: The MBMA established a quantitative relationship between RSV SNA and clinical endpoints. This relationship was quantitatively consistent with animal model challenge experiments and results of a recently published clinical trial. Additionally, SNA elicited by increasing doses of MK-1654 in humans reduced RSV symptomatic infection rates with a quantitative relationship that approximated the MBMA. The MBMA indicated a high probability that a single dose of ≥ 75 mg of MK-1654 will result in prophylactic efficacy (> 75% for 5 months) in infants. INTERPRETATION: An MBMA approach can predict efficacy of neutralizing antibodies against RSV and potentially other respiratory pathogens.