Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Biologicals ; 57: 46-49, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30553568

RESUMO

Insoluble particulate matter test for injections in pharmacopoeia is mandatory for parenteral drug products. In this test using light obscuration, four measurements of at least 5-mL are required. Since therapeutic protein injections of low dosage volumes are getting more popular, reduction of test volumes is desired. In this collaborative study, the impact of lower measurement volume on the accuracy and precision of particle count was evaluated using 2, 5, 10, and 25-µm polystyrene count standards for the validity of test with reduced sample volumes. Good accuracy (3000 particles/mL ±â€¯10%) was obtained at all measurement volumes, and the inter-run variability (RSD) was the same levels between 5 and 1 mL. Although the inter-run variability increased at 0.2 mL, it was below 5%. These results indicated that light obscuration method can be used with 5 mL-0.2 mL, and that it is feasible for monitoring particles ≥2 µm.


Assuntos
Técnicas de Química Analítica/métodos , Contaminação de Medicamentos/prevenção & controle , Estudos de Viabilidade , Material Particulado/análise , Animais , Técnicas de Química Analítica/normas , Humanos , Tamanho da Partícula , Material Particulado/química , Reprodutibilidade dos Testes , Solubilidade
2.
Rapid Commun Mass Spectrom ; 28(8): 921-32, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24623697

RESUMO

RATIONALE: Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes. METHODS: The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs. RESULTS: Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data. CONCLUSIONS: PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products.


Assuntos
Cromatografia Líquida/métodos , Eritropoetina/química , Espectrometria de Massas/métodos , Polissacarídeos/análise , Proteínas Recombinantes/química , Humanos , Análise Multivariada , Polissacarídeos/química , Análise de Componente Principal
3.
Anal Biochem ; 420(1): 61-7, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21951784

RESUMO

Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.


Assuntos
Medição da Troca de Deutério/métodos , Insulina Lispro/química , Insulina de Ação Prolongada/química , Insulina/análogos & derivados , Insulina/análise , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Estabilidade de Medicamentos , Humanos , Injeções Subcutâneas , Insulina Glargina , Insulina Lispro/farmacocinética , Insulina de Ação Prolongada/farmacocinética , Cinética , Dados de Sequência Molecular , Análise de Componente Principal
4.
J Pharm Sci ; 111(3): 648-654, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34619153

RESUMO

Flow imaging (FI) has emerged as a powerful tool to evaluate insoluble particles derived from protein aggregates as an orthogonal method to light obscuration (LO). However, few reports directly compare the FI and LO method in the size and number of protein particles in commercially available therapeutic protein injections. In this study, we measured the number of insoluble particles in several therapeutic protein injections using both FI and LO, and characterized these particles to compare the analytical performance of the methods. The particle counts measured using FI were much higher than those measured using LO, and the difference depended on the products or features of particles. Some products contained a large number of transparent and elongated particles, which could escape detection using LO. Our results also suggested that the LO method underestimates the size and number of silicone oil droplets in prefilled syringe products compared to the FI method. The count of particles ≥10 µm in size in one product measured using FI exceeded the criteria (6000 counts per container) defined in the compendial particulate matter test using the LO method. Thus precaution should be taken when setting the acceptance criteria of specification tests using the FI method.


Assuntos
Material Particulado , Proteínas , Injeções , Tamanho da Partícula
5.
J Pharm Sci ; 111(10): 2745-2757, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35839866

RESUMO

In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles.


Assuntos
Óleos de Silicone , Silicones , Tamanho da Partícula , Proteínas
6.
Biol Pharm Bull ; 34(1): 13-23, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21212511

RESUMO

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.


Assuntos
Exossomos/metabolismo , Proteômica , Saliva/química , Proteínas e Peptídeos Salivares/metabolismo , Biomarcadores , Exossomos/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas e Peptídeos Salivares/análise , Proteínas e Peptídeos Salivares/genética
7.
Biologicals ; 39(3): 171-80, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21549615

RESUMO

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.


Assuntos
Produtos Biológicos/química , Monossacarídeos/análise , Amino Açúcares/análise , Amino Açúcares/normas , Produtos Biológicos/normas , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Cromatografia por Troca Iônica/métodos , Cromatografia por Troca Iônica/normas , Eritropoetina/química , Excipientes , Glicosilação , Monossacarídeos/normas , Proteínas Recombinantes , Padrões de Referência , Reprodutibilidade dos Testes , Ácidos Siálicos/análise , Ácidos Siálicos/normas , Ativador de Plasminogênio Tecidual/química
8.
J Pharm Sci ; 109(5): 1652-1661, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31927040

RESUMO

As reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019. At the 2018 meeting, Dr. Rukman DeSilva of the U.S. Food and Drug Administration and Dr. Srivalli Telikepalli of the National Institute of Standards and Technology participated as guest speakers. At the 2019 meeting, we invited Prof. John Carpenter of the University of Colorado, Prof. Gerhard Winter and Prof. Wolfgang Friess of Ludwig Maximilian University of Munich, and Dr. Tim Menzen of Coriolis Pharma Research, as guest commentators. In both meetings, the main research topic was strategies for the characterization and control of protein aggregates/subvisible particles in drug products. Specifically, the use of the light obscuration method for insoluble particulate matter testing with reduced injection volumes, and a comparison of analytical performance between flow imaging and light obscuration were discussed. Other topics addressed included host cell protein analysis, bioassay, and quality control strategies. In this commentary, the recent achievements of the research group, meeting discussions, and future perspectives are summarized.


Assuntos
Produtos Biológicos , Bioensaio , Fatores Biológicos , Japão , Tamanho da Partícula , Controle de Qualidade
9.
MAbs ; 11(5): 826-836, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30990348

RESUMO

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen-deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs. Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-ß-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen-deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance-solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.


Assuntos
Galactose/química , Polissacarídeos/química , Rituximab/química , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Galinhas/imunologia , Fucose/química , Fucose/imunologia , Galactose/imunologia , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Manose/química , Manose/imunologia , Polissacarídeos/imunologia , Rituximab/metabolismo , Rituximab/uso terapêutico
10.
J Pharm Sci ; 108(2): 832-841, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30121316

RESUMO

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.


Assuntos
Imunoglobulinas Intravenosas/química , Agregados Proteicos , Japão , Luz , Imagem Óptica , Tamanho da Partícula , Tecnologia Farmacêutica
11.
J Chromatogr B Analyt Technol Biomed Life Sci ; 869(1-2): 20-30, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18514042

RESUMO

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/sangue , Glicoproteínas/química , Espectrometria de Massas em Tandem/métodos , Bases de Dados de Proteínas , Glicopeptídeos/análise , Glicopeptídeos/isolamento & purificação , Glicoproteínas/metabolismo , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional
12.
Sci Rep ; 8(1): 3955, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29500371

RESUMO

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.


Assuntos
Anticorpos/uso terapêutico , Polissacarídeos/química , Receptores de IgG/química , Anticorpos/isolamento & purificação , Citotoxicidade Celular Dependente de Anticorpos , Cromatografia Líquida/métodos , Humanos
13.
J Chromatogr A ; 1160(1-2): 263-9, 2007 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-17570377

RESUMO

N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/normas , Cromatografia Líquida/métodos , Ciclotrons , Análise de Fourier , Espectrometria de Massas/métodos , Nanotecnologia/métodos , Ácidos Neuramínicos/análise , Calibragem , Membrana Celular/química , Células HL-60 , Humanos , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Controle de Qualidade , Frações Subcelulares/química
14.
J Pharm Sci ; 106(12): 3431-3437, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28802881

RESUMO

The research and development of next-generation innovative medicines is a prominent interest of both the government and industries in Japan. On June 29, 2017, a kickoff meeting of a new research group focused on the quality issues of biopharmaceuticals was held in Tokyo with Prof. John Carpenter as an invited guest. The group's research focuses mainly on the evaluation and control of protein aggregates/subvisible particles in drug products, but the research topics also include glycan analysis, host-cell protein evaluation, bioassay validation, and analytical quality by design. The purpose of the group's activities is to resolve the critical and fundamental quality issues important to pharmaceutical companies through the collaboration of industries, academia, and regulatory agencies. In this commentary, our current plan to address these issues and the discussion at the kickoff meeting are described.


Assuntos
Fatores Biológicos/química , Biofarmácia/normas , Indústria Farmacêutica/normas , Bioensaio/métodos , Biotecnologia/métodos , Humanos , Japão , Polissacarídeos/química , Proteínas/química , Controle de Qualidade , Pesquisa , Tecnologia Farmacêutica/normas
15.
J Chromatogr A ; 1103(2): 296-306, 2006 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-16364349

RESUMO

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Oligossacarídeos de Cadeias Ramificadas/análise , Antígenos Thy-1/química , Animais , Química Encefálica , Ciclotrons , Análise de Fourier , Glicosilação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Ratos
16.
Nat Commun ; 7: 11205, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27046227

RESUMO

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Autoanticorpos/química , Autoanticorpos/metabolismo , Sequência de Carboidratos , Colágeno Tipo II/imunologia , Colágeno Tipo II/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Dados de Sequência Molecular , Ácidos Siálicos/imunologia , Ácidos Siálicos/metabolismo
17.
J Chromatogr A ; 1094(1-2): 105-17, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16257296

RESUMO

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.


Assuntos
Química Encefálica , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Glicoproteínas/química , Espectrometria de Massas/métodos , Antígenos Thy-1/química , Sequência de Aminoácidos , Animais , Glicosilação , Ratos
18.
MAbs ; 7(6): 1138-50, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26261057

RESUMO

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos CD20/imunologia , Bombyx/genética , Proteínas Recombinantes/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cromatografia Líquida , Proteínas do Sistema Complemento/imunologia , Cricetinae , Cricetulus , Citotoxicidade Imunológica/imunologia , Glicosilação , Humanos , Espectrometria de Massas , Camundongos , Proteínas Recombinantes/metabolismo , Rituximab/genética , Rituximab/imunologia , Rituximab/metabolismo
19.
Toxicol Lett ; 143(2): 233-8, 2003 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-12749827

RESUMO

We previously showed that dibutyltin dichloride (DBTCl) at 7.6 mg/kg and higher on days 0-3 of pregnancy caused implantation failure and a decline in serum progesterone levels in rats and hypothesized that the decline is responsible for the implantation failure. This study was conducted to determine the protective effects of progesterone on the DBTCl-induced implantation failure in rats. Rats were given oral DBTCl at 0, 7.6, or 15.2 mg/kg on days 0-3 of pregnancy and/or subcutaneous progesterone at 2 mg/rat on days 0-8 of pregnancy. The reproductive outcome was determined on day 9 of pregnancy. No effects of administration of progesterone alone on the pregnancy rate and number of implantations were found. The pregnancy rate and number of implantations were significantly decreased after administration of DBTCl alone. The pregnancy rate and number of implantations were higher in the groups given DBTCl and progesterone than the groups given DBTCl alone. The present data indicate that progesterone protects, at least in part, against the DBTCl-induced implantation failure and support our hypothesis that the decline in progesterone levels is a primary mechanism for the implantation failure due to DBTCl.


Assuntos
Implantação do Embrião/efeitos dos fármacos , Compostos Orgânicos de Estanho/farmacologia , Progesterona/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Gravidez , Ratos , Ratos Wistar , Aumento de Peso/efeitos dos fármacos
20.
Reprod Toxicol ; 17(4): 393-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12849849

RESUMO

In our previous study, dibutyltin dichloride (DBTCl) caused preimplantation embryonic loss and postimplantation embryonic loss in rats following administration at 7.6 mg/kg and above on Days 0-3 and at 3.8 mg/kg and above on Days 4-7 of pregnancy, respectively. This study was designed to assess the effects of DBTCl on uterine function as a cause of early embryonic loss using pseudopregnant rats. DBTCl was given orally to pseudopregnant rats at 3.8, 7.6 or 15.2 mg/kg on pseudopregnant day (PPD) 0-3 or on PPD 4-7. The decidual cell response was induced by bilateral uterine scratch on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of DBTCl at 7.6 mg/kg and above on PPD 0-3 and PPD 4-7. DBTCl had no effect on the serum estradiol levels and number of corpora lutea. Administration of progesterone reversed the suppression of uterine decidualization in rats given DBTCl on PPD 0-3. It can be concluded that DBTCl suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to DBTCl exposure.


Assuntos
Decídua/efeitos dos fármacos , Perda do Embrião/etiologia , Compostos Orgânicos de Estanho/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Decídua/patologia , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Perda do Embrião/induzido quimicamente , Feminino , Idade Gestacional , Intubação Gastrointestinal , Tamanho do Órgão/efeitos dos fármacos , Compostos Orgânicos de Estanho/administração & dosagem , Progesterona/sangue , Pseudogravidez/sangue , Ratos , Ratos Wistar
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA