RESUMO
A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Análise de Sequência de Proteína/métodos , Albuminas/química , Aminoácidos/química , Animais , Automação , Bovinos , Cromatografia/métodos , Humanos , Hidrólise , Fragmentos Fab das Imunoglobulinas/química , Proteínas Recombinantes/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Raios Ultravioleta , o-Ftalaldeído/químicaRESUMO
We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)ß(3) . Because α(ν)ß(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.
Assuntos
Complexos de Coordenação/síntese química , Oligopeptídeos/química , Compostos Radiofarmacêuticos/síntese química , Animais , Linhagem Celular Tumoral , Complexos de Coordenação/química , Gadolínio/química , Radioisótopos de Gálio/química , Glioblastoma/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Modelos Moleculares , Oligopeptídeos/metabolismo , Tomografia por Emissão de Pósitrons , Ligação Proteica , Compostos Radiofarmacêuticos/química , Transplante HeterólogoRESUMO
α(V)ß(3) Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near-infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α(V) ß(3) integrin-overexpressing tumors. DA364's binding affinity for α(V)ß(3) integrin was first evaluated in vitro. Human α(V)ß(3) integrin-positive, U-87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α(V)ß(3) integrin on U-87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic-RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α(V)ß(3) integrin-overexpressing tumors.
Assuntos
Carbocianinas , Glioblastoma/diagnóstico , Integrina alfaVbeta3/análise , Peptídeos Cíclicos , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes , Humanos , Camundongos , Camundongos Nus , Técnicas de Sonda Molecular , Transplante de Neoplasias , Peptídeos Cíclicos/química , Tomografia por Emissão de Pósitrons , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição TecidualRESUMO
An immunofluorescence method that allows detection and quantification of recombinant proteins in complex mixtures (e.g. crude cell extracts and fermentation media) is described. The method is based on a non-denaturing chromatographic analysis which is performed after the addition of a fluorescently labelled antibody specific for the target protein. HPLC analysis through a size-exclusion column allows to separate and quantify the immunocomplex and the excess antibody, which are the only fluorescent species in the mixture. The method was applied to the analysis of recombinant human Fabs expressed in bacteria. Preliminary validation data are reported.