RESUMO
High-throughput high-performance liquid chromatography/mass spectrometry can be used in the analysis of high-throughput organic synthesis products, bioanalytical target analysis for preclinical and clinical studies, and early absorption, distribution, metabolism and excretion (ADME) screening. New techniques are emerging, including system automation, faster analysis, programmed multiple extraction and analysis columns, multiple electrospray ionization channels, and automated 96-well sample preparation.
Assuntos
Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos/métodos , Espectrometria de Massas , Tecnologia Farmacêutica/métodos , Patentes como AssuntoRESUMO
A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Preparações Farmacêuticas/química , Cromatografia , Cromatografia Líquida de Alta Pressão , Análise de Injeção de Fluxo , Preparações Farmacêuticas/isolamento & purificaçãoRESUMO
The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.
Assuntos
Eletroforese/métodos , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/química , Acetatos/química , Acrilamidas/química , Adsorção , Sequência de Aminoácidos , Misturas Anfolíticas/química , Animais , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Cabras/imunologia , Soros Imunes/imunologia , Focalização Isoelétrica/métodos , Ponto Isoelétrico , Dados de Sequência Molecular , Neuraminidase/metabolismo , Plasminogênio/metabolismo , Álcool de Polivinil/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Dodecilsulfato de Sódio/química , Tensoativos/química , Ativador de Plasminogênio Tecidual/imunologia , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.