Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Drug Metab Dispos ; 46(4): 451-457, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29386233

RESUMO

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 , Ratos
3.
Bioanalysis ; 8(15): 1611-1622, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27397670

RESUMO

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.


Assuntos
Anticorpos Monoclonais/química , Asparagina/análise , Ácido Aspártico/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Humanos , Imunoprecipitação/métodos , Isomerismo , Macaca fascicularis , Masculino , Espectrometria de Massas em Tandem/métodos
4.
Vaccine ; 34(35): 4250-4256, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27269057

RESUMO

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/genética
5.
Bioanalysis ; 8(4): 265-74, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26807991

RESUMO

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/farmacologia
6.
Hum Vaccin Immunother ; 11(6): 1518-25, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25997113

RESUMO

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Cromatografia Líquida/métodos , Vacinas Meningocócicas/química , Espectrometria de Massas em Tandem/métodos , Descoberta de Drogas/métodos , Humanos , Neisseria meningitidis Sorogrupo B/imunologia
7.
Bioanalysis ; 6(13): 1795-811, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25157486

RESUMO

BACKGROUND: The disease state can modulate the penetration of large antibody-sized therapeutic molecules into affected tissues. Suitable bioanalytical methods are required for the quantitative analysis of drug tissue levels to enable a better understanding of the parameters influencing drug penetration and target engagement. RESULTS: Described is a sensitive and selective LC-MS/MS assay for the quantification of human mAb molecules in mouse tissues. By homogenizing tissues directly into serum, a common serum calibration curve can be used for multiple tissues. A generic procedure was used for affinity enrichment. An analytical range of 20 - 20,000 ng/ml was achieved in serum. CONCLUSION: The method described here can be applied for the quantitative analysis of mAb and Fc-fusion therapeutic molecules in a variety of animal tissue matrices.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/análise , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/metabolismo , Cromatografia de Afinidade , Feminino , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/análise , Análise de Regressão , Pele/metabolismo , Tripsina/metabolismo , Ustekinumab
8.
Anal Chem ; 82(16): 6877-86, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20704377

RESUMO

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/metabolismo
9.
Diabetes ; 55(4): 1034-42, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16567526

RESUMO

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/farmacologia
10.
Rapid Commun Mass Spectrom ; 17(14): 1600-10, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12845586

RESUMO

Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry (ITMS) has been evaluated for automated protein identification. By using signal averaging and long ion-injection times, protein identification limits in the 50-fmol range are achieved for standard protein digests. Data acquisition requires 7.5 min or less per sample and the MS/MS spectra files are automatically processed using the SEQUEST database searching algorithm. AP-MALDI-ITMS was compared with the widely used methods of microLC/MS/MS (ion trap) and automated MALDI-TOF peptide mass mapping. Sample throughput is 10-fold greater using AP-MALDI compared with microcapillary liquid chromatography/tandem mass spectrometry (microLC/MS/MS). The protein sequence coverage obtained from AP-MALDI-MS/MS spectra matched by SEQUEST is lower compared with microLC/MS/MS and MALDI-TOF mass mapping. However, by using the AP-MALDI full-scan peptide mass fingerprint spectrum, sequence coverage is increased. AP-MALDI-ITMS was applied for the analysis of Coomassie blue stained gels and was found to be a useful platform for rapid protein identification.


Assuntos
Proteínas/química , Animais , Autoanálise , Catalase/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Biochemistry ; 41(50): 14734-47, 2002 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-12475222

RESUMO

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.


Assuntos
Peptídeos/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Dissulfetos/química , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA