RESUMO
The glycosylation of proteins is of particular interest in biopharmaceutical applications. The detailed characterization of glycosylation based on the released carbohydrates is mandatory since the protein stability, folding, and efficacy are strongly dependent on the structural diversity inherent in the glycan moieties of a glycoprotein. For glycan pattern analysis, capillary electrophoresis with laser-induced fluorescence using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans is used frequently. In this paper, a robust capillary electrophoresis-mass spectroscopy method both for the analysis of APTS-labeled glycans and unlabeled charged glycans is presented. The background electrolyte consists of 0.7 M ammonia and 0.1 M ε-aminocaproic acid in water/methanol 30:70 (v/v). High separation efficiency including separation of structural isomers was obtained. The method was validated in terms of reproducibility and linearity. Submicromolar sensitivity is achieved with linearity up to 24 µM. The ability to analyze APTS-labeled, as well as unlabeled, charged glycans enables the determination of labeling and ionization efficiency: APTS-labeled glycans show a factor of three better ionization efficiency compared to non-labeled native glycans. The presented method is applied to the analysis of pharmaceutical products. Furthermore, the system can be applied to the analysis of 2-ANSA-labeled glycans, though separation efficiency is limited.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Polissacarídeos/análise , Sequência de Carboidratos , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Espectrometria de Massas/instrumentação , Dados de Sequência Molecular , Pirenos/química , Reprodutibilidade dos TestesRESUMO
The Fc-fusion protein atacicept is currently under clinical investigation for its biotherapeutic application in autoimmune diseases owing to its ability to bind the two cytokines B-Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL). Like typical recombinant IgG-based therapeutics, atacicept is a glycoprotein whose glycosylation-related heterogeneity arises from the glycosylation-site localization, site-specific occupation and structural diversity of the attached glycans. Here, we present a first comprehensive site-specific N- and O-glycosylation characterization of atacicept using mass spectrometry-based workflows. First, N- and O-glycosylation sites and their corresponding glycoforms were identified. Second, a relative quantitation of the N-glycosylation site microheterogeneity was achieved by glycopeptide analysis, which was further supported by analysis of the released N-glycans. We confirmed the presence of one N-glycosylation site, carrying 47 glycoforms covering 34 different compositions, next to two hinge region O-glycosylation sites with core 1-type glycans. The relative O-glycan distribution was analyzed based on the de-N-glycosylated intact protein species. Overall, N- and O-glycosylation were consistent between two individual production batches.
Assuntos
Espectrometria de Massas , Polissacarídeos/análise , Proteínas Recombinantes de Fusão/química , GlicosilaçãoRESUMO
Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated.
Assuntos
Medicamentos Biossimilares/farmacologia , Hormônio Foliculoestimulante Humano/farmacologia , Hormônio Foliculoestimulante/farmacologia , Proteínas Recombinantes/farmacologia , Asparagina/metabolismo , Fucose/metabolismo , Glicopeptídeos/química , Glicosilação/efeitos dos fármacos , Humanos , Ácido N-Acetilneuramínico/metabolismo , Mapeamento de Peptídeos , Polissacarídeos/metabolismo , Subunidades Proteicas/metabolismo , Padrões de ReferênciaRESUMO
A sesquiterpene and a tetrahydroxylated acyclic diterpene as well as two known monoterpenes, 6 C(13)nor-terpenes and 11 aromatic compounds were isolated from the water extract of Malva silvestris. The structures of the compounds were determined by spectroscopic NMR and MS analysis. Effects of these compounds on germination and growth of dicotyledon Lactuca sativa L. (lettuce) were studied in the 10(-4)-10(-7)M concentration range.
Assuntos
Malva/química , Fenóis/química , Terpenos/química , Lactuca/efeitos dos fármacos , Lactuca/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Fenóis/farmacologia , Componentes Aéreos da Planta/química , Solventes/química , Terpenos/farmacologia , Água/químicaRESUMO
Five C13 norisoprenoids, one of them glucosilated, have been isolated from the leaves of Brassica fruticulosa (Brassicaceae). The structure (3S,4R,7E,9S)-3,4,9-trihydroxy-5,7-megastigmadiene, has been assigned to the new compound. All the structures have been determined by spectroscopic means and chemical correlations.
Assuntos
Brassica/química , Norisoprenoides/isolamento & purificação , Espectroscopia de Ressonância MagnéticaRESUMO
Seven cinnamic acid amides have been isolated from Chenopodium album. The structures have been attributed by means of their spectral data. One of them, N-trans-4-O-methylferuloyl 4'-O-methyldopamine, is described for the first time. Their effects on germination and growth of dicotyledons Lactuca sativa L. (lettuce) and Lycopersicon esculentum L. (tomato) and of monocotyledon Allium cepa L. (onion) as standard target species have been studied in the range concentration 10(-4)-10(-7) M.
Assuntos
Amidas/química , Amidas/farmacologia , Chenopodium album/química , Cinamatos/química , Germinação/efeitos dos fármacos , Verduras/efeitos dos fármacos , Amidas/isolamento & purificação , Cinamatos/isolamento & purificação , Cinamatos/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Parabenos/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Verduras/crescimento & desenvolvimentoRESUMO
Five lignans, five neolignans, two sesquilignans, and a dilignan were identified from a phytotoxic extract of Brassica fruticulosa L. Compounds 8, 9, 12, and 13 have been isolated for the first time. Structures were determined on the basis of their spectroscopic features. Their effects on the germination and growth of two dicotyledons, Lactuca sativa (lettuce) and Lycopersicon esculentum (tomato), and a monocotyledon, Allium cepa (onion), as standard target species have been studied.
Assuntos
Brassica/química , Germinação/efeitos dos fármacos , Lignanas/farmacologia , Desenvolvimento Vegetal , Lactuca/efeitos dos fármacos , Lactuca/crescimento & desenvolvimento , Lignanas/química , Lignanas/isolamento & purificação , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Cebolas/efeitos dos fármacos , Cebolas/crescimento & desenvolvimento , Extratos Vegetais/químicaRESUMO
The roots of Chenopodium album were infused in MeOH, and the extract was partitioned between AcOEt and H2O. AcOEt-Soluble material was subjected to different silica-gel column chromatographies and then purified by reverse-phase HPLC to afford a new cinnamic acid amide alkaloid as a racemic mixture. The new compound, named chenoalbicin (1), was characterized by extensive spectroscopic investigation, especially 1D and 2D NMR spectroscopy. Its effects on the germination and growth of Lactuca sativa L. has been studied. The results are reported as percentage differences of germination, root elongation, and shoot elongation from the control at concentrations ranging from 10(-4) to 10(-7) M.
Assuntos
Alcaloides/química , Amidas/química , Chenopodium album , Cinamatos/química , Alcaloides/isolamento & purificação , Amidas/isolamento & purificação , Cinamatos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Raízes de Plantas , Sementes/crescimento & desenvolvimentoRESUMO
Antioxidant capacity of the aqueous extract of Malva sylvestris was measured by its ability to scavenge the DPPH and superoxide anion radicals and to induce the formation of a phosphomolybdenum complex. Analysis of the extract, carried out by different chromatographic techniques, led to the isolation of eleven compounds: 4-hydroxybenzoic acid, 4-methoxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid, ferulic acid, methyl 2-hydroxydihydrocinnamate, scopoletin, N-trans-feruloyl tyramine, a sesquiterpene, (3R,7E)-3-hydroxy-5,7-megastigmadien-9-one, and (10E,15Z)-9,12,13-trihydroxyoctadeca-10,15-dienoic acid. The antioxidant activities of all these compounds are reported.
Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Malva/química , Compostos de Bifenilo/química , Indicadores e Reagentes , Itália , Picratos/química , Espécies Reativas de Oxigênio/química , Espectrofotometria UltravioletaRESUMO
Cinnamic acid, 4-hydroxy-cinnamic acid, ferulic acid, methyl ferulate, sinapic acid, methyl 3-(4-hydroxy-3-methoxyphenyl)propanoate, 4-(1-hydroxyethyl)-2-methoxyphenol, vanillyl alcohol, 4-(hydroxymethyl)-2-methoxyphenol, 4-hydroxy-3-methoxybenzoic acid, 4-vinylphenol, 4-methylbenzaldehyde, N-[2-(1H-indol-3-yl)ethyl]acetamide, pinoresinol, syringaresinol, lariciresinol, 5,5'-dimethoxy-lariciresinol, threo-guaiacylglycerol-3-beta-4-syringaresinol ether and two new sesquilignans, namely, threo-guaiacylglycerol-alpha-O-methyl-beta-O-4-syringaresinol ether and threo-syringylglycerol-alpha-O-methyl-beta-O-4-syringaresinol ether, were isolated and identified as components of Chenopodium album. Constitutions were established on the basis of spectroscopic data, including two-dimensional NMR analyses.