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1.
Clin Chem ; 66(2): 282-301, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32040572

RESUMO

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.

3.
Bioanalysis ; 9(20): 1573-1588, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29072496

RESUMO

AIM: IP-10 is a protein target for the treatment of Crohn's disease. Inhibition of IP-10 by anti-IP-10 mAbs neutralizes its various biological activities. The measurement of free IP-10 suppression as a target engagement biomarker is required for the assessment of drug effect on the target. RESULTS: The development of highly sensitive immunoaffinity-LC-MS/MS assays for quantifying free and total IP-10 in cynomolgus monkey serum is reported for the first time. This paper details strategies for maximizing assay sensitivity by selecting digestion routes, and optimizing immunocapture to achieve full recovery and minimal matrix effect. For the free IP-10 assay, bioanalytical strategies have been established to minimize drug/ligand dissociation. CONCLUSION: The assays have been implemented for target engagement measurement, pharmacokinetic-pharmacodynamic correlation, and human dose projections.


Assuntos
Biomarcadores/sangue , Quimiocina CXCL10/sangue , Cromatografia de Afinidade , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Cromatografia Líquida de Alta Pressão , Humanos , Ligantes , Macaca fascicularis , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
4.
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
5.
Bioanalysis ; 8(13): 1383-401, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27277879

RESUMO

BACKGROUND: Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. METHODS: A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. RESULTS & CONCLUSION: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.


Assuntos
Imunoconjugados/sangue , Preparações Farmacêuticas/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Haplorrinos , Humanos , Imunoensaio/métodos , Imunoconjugados/análise , Limite de Detecção , Preparações Farmacêuticas/análise , Ratos
6.
Anal Biochem ; 503: 71-8, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27033006

RESUMO

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.


Assuntos
Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Marcação por Isótopo , Metabolômica , Animais , Ácidos e Sais Biliares/química , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , Ratos
7.
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
8.
Bioanalysis ; 8(3): 193-204, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811930

RESUMO

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.


Assuntos
Cromatografia Líquida/métodos , Miostatina/análise , Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/economia , Análise Custo-Benefício , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miostatina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Espectrometria de Massas em Tandem/economia , Tripsina/metabolismo
9.
Bioanalysis ; 7(19): 2501-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26466806

RESUMO

The analysis of endogenous and exogenous analytes in biological matrices presents several challenges to the bioanalyst. These analytes are often present at low concentrations, typically in complex matrices, and may have physicochemical properties that are not amenable to LC-MS analysis. The bioanalyst thus relies heavily on the formation of analyte derivatives for the efficient quantification of these compounds. These derivatives are also critically employed to derive information on the biology of living systems, potential drug or disease targets, and biomarkers of drug efficacy, safety, or disease progression. In this perspective, we demonstrate how analyte derivatives are applied in modern bioanalytical workflows and we discuss the potential use of these derivatives in the future.


Assuntos
Biomarcadores/análise , Preparações Farmacêuticas/análise , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Humanos , Marcação por Isótopo , Preparações Farmacêuticas/química
10.
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
11.
Bioanalysis ; 6(13): 1827-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25157488

RESUMO

Both LBAs and LC-MS-based assays are reviewed and summarized for applications in quantitative protein analysis. A strategy for platform selection is proposed based on several factors that contribute to the complexities of bioanalysis of biologics. Protein types, multiple co-existing forms, post-translational modifications, and affinities to ADA, targets, and endogenous proteins need to be considered when selecting the most appropriate platform. Other factors, such as intended use of data, assay sensitivity, available reagents, and multiple analytes also impact on the choice of bioanalytical platform. At BMS, strategies for the seamless integration of both platforms are being implemented to provide not only PK/PD data of the molecules but also useful information of the amino acid structure and functional relationship of the proteins.


Assuntos
Cromatografia Líquida de Alta Pressão , Ligantes , Proteínas/análise , Espectrometria de Massas em Tandem , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Leptina/análise , Leptina/farmacocinética , Proteínas/isolamento & purificação , Proteínas/farmacocinética
12.
Bioanalysis ; 6(14): 1901-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25158962

RESUMO

BACKGROUND: (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, also known as LY379268, a group II metabotropic glutamate receptor agonist, has been widely used in neuroscience as a model compound in studies evaluating antipsychotic drugs for the treatment of schizophrenia. MATERIALS & METHODS: So far, no reports describing methods of the bioanalysis of LY379268 have been published. Here, a novel method is presented for determining LY379268 in rat plasma employing precolumn derivatization with pentafluorobenzoyl chloride reagent coupled to liquid chromatography/mass spectrometry. CONCLUSION: Chemical derivatization of a low-molecular-weight and highly polar molecule yields a derivative that is retained on a reversed-phase liquid chromatography column with improved tandem mass spectrometric response.


Assuntos
Aminoácidos/sangue , Compostos Bicíclicos Heterocíclicos com Pontes/sangue , Cromatografia Líquida/métodos , Receptores de Glutamato Metabotrópico/agonistas , Espectrometria de Massas em Tandem/métodos , Aminoácidos/química , Animais , Benzoatos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Cromatografia de Fase Reversa/métodos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Ratos
13.
Rapid Commun Mass Spectrom ; 28(13): 1535-43, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24861605

RESUMO

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS: The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). RESULTS: Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. CONCLUSIONS: The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.


Assuntos
Quimiocina CXCL10/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/química , Quimiocina CXCL10/química , Formiatos , Humanos , Análise dos Mínimos Quadrados , Sensibilidade e Especificidade , Tripsina
14.
Anal Biochem ; 452: 10-2, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24534252

RESUMO

L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS).


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fosfosserina/líquido cefalorraquidiano , Fosfosserina/química , Compostos Organofosforados/química , Succinimidas/química
15.
Rapid Commun Mass Spectrom ; 27(16): 1882-6, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23857934

RESUMO

RATIONALE: Research on disorders of the central nervous system (CNS) has shown that an imbalance in the levels of specific endogenous neurotransmitters may underlie certain CNS diseases. These alterations in neurotransmitter levels may provide insight into pathophysiology, but can also serve as disease and pharmacodynamic biomarkers. To measure these potential biomarkers in vivo, the relevant sample matrix is cerebrospinal fluid (CSF), which is in equilibrium with the brain's interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Accurate analysis of these potential biomarkers can be challenging due to low CSF sample volume, low analyte levels, and potential interferences from other endogenous compounds. METHODS: A protocol has been established for effective method development of bioanalytical assays for endogenous compounds in CSF. Database searches and standard-addition experiments are employed to qualify sample preparation and specificity of the detection thus evaluating accuracy and precision. RESULTS: This protocol was applied to the study of the histaminergic neurotransmitter system and the analysis of histamine and its metabolite 1-methylhistamine in rat CSF. CONCLUSIONS: The protocol resulted in a specific and sensitive novel method utilizing pre-column derivatization ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is also capable of separating an endogenous interfering compound, identified as taurine, from the analytes of interest.


Assuntos
Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida de Alta Pressão/métodos , Histamínicos/líquido cefalorraquidiano , Histamina/líquido cefalorraquidiano , Metilistaminas/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Animais , Ratos
16.
Bioanalysis ; 4(16): 2059-65, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22946921

RESUMO

In the last several years, dried blood spot (DBS) sampling has re-emerged and attracted a great interest in the pharmaceutical industry as a microsampling technology for drug discovery and development studies. Although significant progress has been made to understand strengths and weaknesses of the technique, many organizations are still at the evaluation stage and experimental observations have resulted in more questions being raised as to whether there is a real future for this technology in pharmaceutical research, especially in support of pharmacokinetic studies. This article summarizes recently gained knowledge against the originally projected advantages of this technique, discusses some practical challenges that need to be overcome before DBS can be widely applied in drug development studies, and highlights some specific study types where DBS can be applied with a good benefit:risk ratio. The authors hope this article can stimulate further discussions about what are the next steps for DBS.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/tendências , Preparações Farmacêuticas/sangue , Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Farmacocinética , Plasma/química , Ligação Proteica , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
17.
Bioanalysis ; 4(9): 1057-64, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22612686

RESUMO

BACKGROUND & METHOD: The small sample volumes characteristic to dried blood spot (DBS) sampling enabled us to right-shift the linear dynamic range of an LC-MS/MS plasma assay tenfold and eliminate the need for extensive sample dilution in support of three discovery toxicology studies in which both plasma and DBS samples were collected. With the right-shifted DBS assay range, no DBS study samples required dilution, while all of the plasma samples were diluted 5-50-fold. RESULTS: DBS standard curves from 78-80,000 nM were linear, the performance of the curve and QC samples was within acceptable discovery-assay criteria and individual plasma and DBS data were comparable. Linear correlations of C(max) and AUC derived from DBS and plasma data resulted in R(2) > 0.9. CONCLUSION: This bioanalytical strategy represents a benefit to the bioanalyst that can expedite the return of data and minimize the potential for error and variability that can result from extensive dilutions of study samples.


Assuntos
Cromatografia Líquida de Alta Pressão/normas , Teste em Amostras de Sangue Seco/normas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Espectrometria de Massas/normas , Plasma/química , Administração Oral , Animais , Área Sob a Curva , Cães , Macaca fascicularis , Preparações Farmacêuticas/análise , Controle de Qualidade , Ratos
18.
Bioanalysis ; 4(5): 511-28, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22409550

RESUMO

BACKGROUND: UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies. MATERIAL & METHODS: Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article. The scope of this work is to demonstrate the potential of Q-TOF for the analysis of typical small molecules, therapeutic peptides (molecular weight <6000 Da), and enzymatically (i.e., trypsin, chymotrypsin and pepsin) cleaved peptides from larger proteins. RESULTS & DISCUSSION: This work focuses mainly on full-scan TOF data obtained under ESI conditions, the major mode of TOF operation in discovery bioanalytical research, where the compounds are selected based on their pharmacokinetic/pharmacodynamic behaviors using animal models prior to selecting a few desirable candidates for further development. Finally, important emerging TOF technologies that could potentially benefit bioanalytical research in the semi-quantification of metabolites without synthesized standards are discussed. Particularly, the utility of captive spray ionization coupled with TripleTOF 5600 was evaluated for improving sensitivity and providing normalized MS response for drugs and their metabolites. The workflow proposed compromises neither the efficiency, nor the quality of pharmacokinetic data in support of early drug discovery programs.


Assuntos
Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Espectrometria de Massas , Cafeína/análise , Cafeína/química , Peptídeos/análise , Peptídeos/química , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Software
19.
Bioanalysis ; 4(1): 29-40, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22191592

RESUMO

BACKGROUND: There is considerable interest in the pharmaceutical industry today in both development of therapeutic proteins as viable biopharmaceutical agents as well as the implementation of microsampling techniques, such as dried blood spots (DBS), as an alternative to current sample collection and handling procedures for biological samples generated in drug discovery and development studies. We have demonstrated that these two techniques can be integrated by developing bioanalytical methods that simultaneously determine the concentrations of unique therapeutic protein constructs, using LC-MS-based detection of multiple surrogate peptides following direct trypsin digestion of DBS. RESULTS: Bioanalytical methods were developed for the simultaneous determination of two structurally different therapeutic proteins (PEGylated-Adnectin™-1, MW 11,144 amu and an Fc-fusion protein, MW 67,082 amu) in a single DBS sample using LC-MS-based detection of multiple peptides generated from different regions of the proteins following trypsin digestion. The same methodology was applied to the analysis of DBS samples collected following dosing of a third unique protein (PEGylated-Adnectin-2) to mice. Although these initial DBS methods were slightly less sensitive than those developed specifically for each individual protein in plasma or serum, the generic digestion procedure yielded sufficient accuracy, precision and an extended linear dynamic range to justify their further evaluation in pharmacokinetic, pharmacodynamic and toxicological studies of selected therapeutic proteins following dosing in preclinical discovery studies. Additionally, DBS samples may offer a convenient, generic platform approach for direct enzymatic digestion and sample preparation for LC-MS-based quantitation of proteins. DBS samples prepared for two of the therapeutic proteins were also stable for at least 2 weeks when stored at room temperature. CONCLUSION: Although the same clarification and interpretation of DBS results will be required (e.g., blood vs plasma levels, hematocrit effects on DBS determinations and red blood cell partitioning) as for small-molecules, there still remains the potential to further develop and expand this strategy with appropriate proteins of interest. While additional studies will be required to validate this approach in specific applications, we have demonstrated the feasibility of using DBS sampling to directly quantify structurally different types of therapeutic proteins in blood in discovery studies and present the potential to simultaneously measure other proteins, such as biomarkers, to augment and integrate data generated from in vivo studies.


Assuntos
Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodos , Peptídeos/sangue , Espectrometria de Massas em Tandem/métodos , Tripsina/química , Animais , Humanos , Camundongos , Tripsina/metabolismo
20.
Rapid Commun Mass Spectrom ; 25(22): 3427-35, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-22002697

RESUMO

Highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods have been developed and implemented for the quantitative determination of a number of peptides under evaluation in our Glucagon-Like Peptide-1 (GLP-1) discovery program for the treatment of diabetes. These peptides are GLP-1 receptor agonists. Due to the high potency, low dose, and low exposure of these peptides, LC/MS/MS-based methods with Lower Limits of Quantitation (LLOQs) (low picomolar range) were required to support discovery pharmacokinetic/ pharmacodynamic (PK/PD) studies. Compared with small molecules, many of these peptides posed significant bioanalytical challenges in the development of highly sensitive methods because of their parent signal splitting as a result of the formation of multiply charged states, the unfavorable fragmentation patterns for Selected Reaction Monitoring (SRM) transitions due to the generation of a large number of small mass product ions with relative low intensities, and adsorption issues observed during sample preparation. This paper details the strategies developed to maximize the sensitivity and improve LLOQs from aspects of mass spectrometry, chromatography, and sample preparation. A LLOQ of 10 picomolar was achieved for all of the investigated peptides using 100 µL of mouse plasma. This is a 100-fold improvement on LLOQs over generic LC/MS/MS-based methods when the same sample volume and the same mass spectrometer platform were used. The methods have been implemented in the support of discovery PK/PD studies.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeos/química , Receptores de Glucagon/agonistas , Espectrometria de Massas em Tandem/métodos , Adsorção , Animais , Calibragem , Cromatografia Líquida de Alta Pressão/normas , Descoberta de Drogas , Receptor do Peptídeo Semelhante ao Glucagon 1 , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/normas
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