RESUMO
Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Inclusão em Parafina , Anticorpos Monoclonais/farmacocinética , Formaldeído/química , Fixação de TecidosRESUMO
Fragment crystallizable (Fc) fusion is commonly used for extending the half-life of biotherapeutics such as cytokines. In this work, we studied the pharmacokinetics of Fc-fused interleukin-10 (IL-10) proteins that exhibited potent antitumor activity in mouse syngeneic tumor models. At pharmacologically active doses of ≥0.1 mg/kg, both mouse Fc-mouse IL-10 and human Fc-human IL-10, constructed as the C terminus of the Fc domain fused with IL-10 via a glycine-serine polypeptide linker, exhibited nonlinear pharmacokinetics after intravenous administration to mice at the doses of 0.05, 0.5, and 5 mg/kg. With a nominal dose ratio of 1:10:100; the ratio of the area under the curve for mouse Fc-mouse IL-10 and human Fc-human IL-10 was 1:181:1830 and 1:75:633, respectively. In contrast, recombinant mouse or human IL-10 proteins exhibited linear pharmacokinetics in mice. Compartmental analysis, using the Michaelis-Menten equation with the in vitro IL-10 receptor alpha binding affinity inputted as the Km, unified the pharmacokinetic data across the dose range. Additionally, nontarget-mediated clearance estimated for fusion proteins was â¼200-fold slower than that for cytokines, causing the manifestation of target-mediated drug disposition (TMDD) in the fusion protein pharmacokinetics. The experimental data generated with a mouse IL-10 receptor alpha-blocking antibody and a human Fc-human IL-10 mutant with a reduced receptor binding affinity showed significant improvements in pharmacokinetics, supporting TMDD as the cause of nonlinearity. Target expression and its effect on pharmacokinetics must be determined when considering using Fc as a half-life extension strategy, and pharmacokinetic evaluations need to be performed at a range of doses covering pharmacological activity. SIGNIFICANCE STATEMENT: Target-mediated drug disposition can manifest to affect the pharmacokinetics of a fragment crystallizable (Fc)-fused cytokine when the nontarget-mediated clearance of the cytokine is decreased due to neonatal Fc receptor-mediated recycling and molecular weight increases that reduce the renal clearance. The phenomenon was demonstrated with interleukin-10 Fc-fusion proteins in mice at pharmacologically active doses. Future drug designs using Fc as a half-life extension approach for cytokines need to consider target expression and its effect on pharmacokinetics at relevant doses.
Assuntos
Interleucina-10 , Animais , Meia-Vida , Humanos , Interleucina-10/farmacocinética , Camundongos , Receptores de Interleucina-10 , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.
Assuntos
Aptâmeros de Nucleotídeos/química , Carcinoma Hepatocelular/diagnóstico , Glipicanas/análise , Imunoensaio , Neoplasias Hepáticas/diagnóstico , Cromatografia Líquida , Humanos , Proteínas Recombinantes/análise , Solubilidade , Espectrometria de Massas em TandemRESUMO
Therapeutic biologics have become a fast-growing segment within the pharmaceutical industry during the past 3 decades. Although the metabolism of biologics is more predictable than small molecule drugs, biotransformation can significantly affect the activity of biologics. Unfortunately, there are only a limited number of published studies on the biotransformation of biologics, most of which are focused on one or a few types of modifications. In this study, an untargeted LC-MS-based differential analysis approach was developed to rapidly and precisely determine the universal biotransformation profile of biologics with the assistance of bioinformatic tools. A human monoclonal antibody (mAb) was treated with t-butyl hydroperoxide and compared with control mAb using a bottom-up proteomics approach. Thirty-seven types of post-translational modifications were identified, and 38 peptides were significantly changed. Moreover, although all modifications were screened and detected, only the ones related to the treatment process were revealed by differential analysis. Other modifications that coexist in both groups were filtered out. This novel analytical strategy can be effectively applied to study biotransformation-mediated protein modifications, which will streamline the process of biologic drug discovery and development.
Assuntos
Produtos Biológicos/química , Biotransformação/fisiologia , Cromatografia Líquida/métodos , Proteínas/química , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/química , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Humanos , Peptídeos/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica/métodos , RatosRESUMO
RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Gangliosídeo G(M1)/análogos & derivados , Neoplasias Pulmonares/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/análise , Biomarcadores/sangue , Biomarcadores/química , Gangliosídeo G(M1)/sangue , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/isolamento & purificação , Humanos , RatosRESUMO
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. 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 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with this NEW Regulation" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication covers the recommendations on Mass Spectrometry Assays, Regulated Bioanalysis/BMV (Part 1A) and Regulatory Inputs (Part 1B). Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 7 and 8 (2024), respectively.
Assuntos
Proteômica , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Biomarcadores/análise , Estados Unidos , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Cromatografia/métodos , BrancosRESUMO
LC-MS analysis of therapeutic antibodies and other biotherapeutics from in-life studies (e.g., serum/plasma) has evolved from simple peptide digestion to peptide mapping and intact mass monitoring. From more advanced analytical approaches, a deeper understanding as to the fate of the biotherapeutic in vivo is gained. Here, we examine the next generation of approaches to facilitate the most comprehensive understanding of large molecule drug fate in circulation. Three case studies are presented: (1) use of relative and absolute calibration curves for biotherapeutic quantitation from the same sample set; (2) top-down mass spectrometry applied to bioanalytical assays; (3) biotherapeutic protein complexes from serum analyzed by native protein MS. We anticipate that these approaches will be further adapted and applied by other research groups.
Assuntos
Proteínas , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas/análise , Peptídeos , Mapeamento de Peptídeos/métodosRESUMO
Time-of-flight MS systems for biopharmaceutical and protein characterization applications may play an even more pivotal role in the future as biotherapeutics increase in drug pipelines and as top-down MS approaches increase in use. Here, a recently developed TOF MS system is examined for monoclonal antibody (mAb) characterization from serum samples. After immunocapture, purified drug material spiked into monkey serum or dosed for an in-life study is analyzed by top-down MS. While characterization aspects are a distinct advantage of the MS platform, MS system and software capabilities are also shown regarding intact protein quantitation. Such applications are demonstrated to help enable comprehensive protein molecule quantitation and characterization by use of TOF MS instrumentation.
Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Elétrons , SoftwareRESUMO
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.
Assuntos
Cromatografia , Vacinas , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Espectrometria de Massas , Oligonucleotídeos , TecnologiaRESUMO
Sensitive, multiplexed protein quantification remains challenging despite recent advancements in LC-MS assays for targeted protein biomarker quantification. High-sensitivity protein biomarker measurements usually require immuno-affinity enrichment of target protein; a process which is highly dependent on capture reagent and limited in capability to measure multiple analytes. Herein, we report a novel antibody-free platform, which measures multiple biomarkers from complex matrices employing a strategically optimized solid-phase extraction cleanup and orthogonal multidimensional LC-MS. Eight human protein biomarkers with different specifications were spiked into canine plasma as a model investigation system. The developed strategy achieved the desired sensitivity, robustness, and throughput via the following steps: (1) post digestion mixed-mode cation exchange-reverse phase SPE enrichment cleaned up the sample initially; (2) rapid, high-pH peptide fractionation further eliminated background components efficiently while selectively enriched signature peptides (SP) to provide sufficient sensitivity for multiple targets; and (3) trapping-micro-LC-MS analysis delivered high sensitivity comparable to a nano-LC-MS method but with much better robustness and throughput for the final analysis. Compared with a conventional LC-MS assay with direct protein digestion and limited clean-up, analysis with this antibody-free platform improved the LLOQ by 1-2 orders of magnitude for the eight protein biomarkers, reaching as low as 5 ng/mL in plasma, with feasible robustness and throughput. This platform was applied for the quantification of biomarkers of respiratory conditions in patients with various lung diseases, demonstrating real-world applicability.
Assuntos
Proteínas , Extração em Fase Sólida , Animais , Anticorpos , Biomarcadores/análise , Cromatografia Líquida/métodos , Cães , Humanos , Espectrometria de Massas/métodos , Peptídeos , Extração em Fase Sólida/métodosRESUMO
Pharmacokinetic/pharmacodynamic (PK/PD) modeling was performed to quantitatively integrate preclinical pharmacology and toxicology data for determining the therapeutic index (TI) of an interleukin-10 (IL-10) fragment crystallizable (Fc) fusion protein. Mouse Fc fused with mouse IL-10 (mFc-mIL-10) was studied in mice for antitumor efficacy, and the elevation of interleukin-18 (IL-18) was examined as a PD biomarker. The in vivo mFc-mIL-10 EC50 for the IL-18 induction was estimated to be 2.4 nM, similar to the in vitro receptor binding affinity (Kd) of 3.2 nM. The IL-18 induction was further evaluated in cynomolgus monkeys, where the in vivo induction EC50 by a human IL-10 human Fc-fusion protein (hFc-hIL-10) was 0.08 nM vs. 0.3 nM measured as the in vitro Kd. The extent of the IL-18 induction correlated with mouse antitumor efficacy and was used to connect mouse efficacy to that in monkeys. The PD-based efficacious dose projected in monkeys was comparable to the results obtained using a PK-based method in which mouse efficacious exposure was targeted and corrected for affinity differences between the species. Furthermore, PK/PD relationships were developed for anemia and thrombocytopenia in monkeys treated with hFc-hIL-10, with thrombocytopenia predicted to be dose-limiting toxicity. Using quantitative pharmacology and toxicology information obtained through modeling work in the same species, the TI of hFc-hIL-10 in monkeys was determined to be 2.4 (vs. PD-based efficacy) and 1.2-3 (vs. PK-based efficacy), indicating a narrow safety margin. The model-based approaches were proven valuable to the developability assessment of the IL-10 Fc-fusion protein.
RESUMO
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th 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 biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.
Assuntos
Vesículas Extracelulares , Vacinas , Biomarcadores/análise , Terapia Baseada em Transplante de Células e Tecidos , Vesículas Extracelulares/química , Humanos , Espectrometria de Massas/métodos , NanomedicinaRESUMO
Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Proteínas/farmacocinética , Animais , Biotransformação , Cromatografia de Afinidade/métodos , Cromatografia Líquida , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunoconjugados/análise , Espectrometria de Massas/economia , Espectrometria de Massas/instrumentação , Proteínas/isolamento & purificação , Manejo de EspécimesRESUMO
Background: Surrogate monoclonal antibodies (mAbs) used in preclinical in vivo studies can be challenging to quantify due to lack of suitable immunoaffinity reagents or unavailability of the mAb protein sequence. Generic immunoaffinity reagents were evaluated to develop sensitive LC-MS/MS assays. Peptides of unknown sequence can be used for selective LC-MS quantification. Results: anti-mouse IgG1 was found to be an effective immunoaffinity reagent, enabling quantification of mouse IgG1 mAbs in mouse serum. Selective peptides of unknown sequence were applied for multiplex LC-MS quantification of two rat mAbs co-dosed in mouse. Conclusion: Generic anti-mouse IgG subtype-specific antibodies can be used to improve assay sensitivity and peptides of unknown sequence can be used to quantify surrogate mAbs when the mAb protein sequence in unavailable.
Assuntos
Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Humanos , Camundongos , RatosRESUMO
Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.
Assuntos
Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/sangue , Polietilenoglicóis/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia de Afinidade/métodos , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Tripsina/metabolismoRESUMO
Delayed-rectifier K+ currents (I(DR)) in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltage-gated K+ channel, K(V)2.1, is expressed in beta-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I(DR) in rodent beta-cells. A novel peptidyl inhibitor of K(V)2.1/K(V)2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration approximately 1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to beta-cell physiology. In mouse beta-cells, GxTX-1 inhibits 90% of I(DR) and, as for K(V)2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the beta-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the beta-cell I(DR), which may provide advantages over currently used therapies for the treatment of type 2 diabetes.
Assuntos
Canais de Potássio de Retificação Tardia/fisiologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Sequência de Aminoácidos , Animais , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/química , Venenos de Aranha/química , Venenos de Aranha/farmacologiaRESUMO
The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies' Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.
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Biomarcadores/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Peptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Conferências de Consenso como Assunto , Guias como Assunto , Ligantes , Bibliotecas de Moléculas Pequenas/químicaRESUMO
BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.
Assuntos
Análise Química do Sangue/métodos , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Isoquinolinas/análise , Limite de Detecção , Oligopeptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Feminino , Humanos , Proteínas Imobilizadas/antagonistas & inibidores , Proteínas Imobilizadas/química , Proteínas Inibidoras de Apoptose/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Engenharia Genética , Imunogenicidade da Vacina , Vacinas Meningocócicas/imunologia , Vesículas Transportadoras/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Macaca mulatta , Vacinas Meningocócicas/genética , Neisseria meningitidis , Neisseria meningitidis Sorogrupo B , Porinas/genéticaRESUMO
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.