Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.212
Filtrar
1.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 871-879, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223005

RESUMO

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Assuntos
Proteínas de Escherichia coli , Peptídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
2.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 1-17, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31128359

RESUMO

Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.


Assuntos
Anticorpos Monoclonais , Eletroforese Capilar , Imunoconjugados , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Humanos , Imunoconjugados/análise , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Focalização Isoelétrica , Espectrometria de Massas
3.
Emerg Microbes Infect ; 8(1): 749-759, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130109

RESUMO

The Zika virus (ZIKV) outbreak and its link to microcephaly triggered a public health concern. To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 heavy and light chain-paired monoclonal antibodies (mAbs) that bind to ZIKV envelope (E) proteins isolated from memory B cells of a ZIKV-infected patient. Three mAbs (7B3, 1C11, and 6A6) that showed the most potent and broad neutralization activities against the African, Asian, and American strains were selected for further analysis. mAb 7B3 showed an IC50 value of 11.6 ng/mL against the circulating American strain GZ02. Epitope mapping revealed that mAbs 7B3 and 1C11 targeted residue K394 of the lateral ridge (LR) epitope of the EDIII domain, but 7B3 has a broader LR epitope footprint and recognizes residues T335, G337, E370, and N371 as well. mAb 6A6 recognized residues D67, K118, and K251 of the EDII domain. Interestingly, although the patient was seronegative for DENV infection, mAb 1C11, originating from the VH3-23 and VK1-5 germline pair, neutralized both ZIKV and DENV1. Administration of the mAbs 7B3, 1C11, and 6A6 protected neonatal SCID mice infected with a lethal dose of ZIKV. This study provides potential therapeutic antibody candidates and insights into the antibody response after ZIKV infection.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Imunização Passiva , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Adulto , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , China , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/isolamento & purificação , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos SCID , Testes de Neutralização , Análise de Sobrevida , Resultado do Tratamento , Infecção por Zika virus/imunologia
4.
J Chromatogr A ; 1599: 152-160, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31084900

RESUMO

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates enabling much higher loadings. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molecular weight aggregates. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggregates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica , Anticorpos Monoclonais/química , Cátions/química , Concentração de Íons de Hidrogênio
5.
Artigo em Inglês | MEDLINE | ID: mdl-31128526

RESUMO

Activated carbon (AC) is a porous solid with a larger surface area and lower cost than chromatography resins. AC has been used in the field of biopharmaceutical manufacturing for plasma-derived products and recombinant monoclonal antibodies (mAb). In our previous study, AC was employed in the purification process of therapeutic mAb as a replacement for Protein A affinity chromatography (PrA). In addition, we designed an all flow-through purification process using AC. In these investigations, greater effective clearance of high-molecular-weight species (HMW), low-molecular-weight species (LMW), host cell proteins (HCP), and DNA was observed compared to that of the conventional Protein A platform purification process. However, it was revealed that mAb recovery from the AC step was lower than that from the PrA step. In this work, to improve mAb recovery from the AC step while maintaining the effective removal of impurities, a pretreatment procedure conducted prior to the AC treatment was investigated. We found that both an ultrafiltration/dilution and reduction in the conductivity of the filtered cell culture supernatant after acid precipitation could improve both the impurity clearance and mAb recovery from the AC treatment. From the obtained results, we designed a two-step purification process in which AC treatment is followed by either cation exchange column chromatography or anion exchange column chromatography, and we compared this against the Protein A platform purification process. Excellent impurity clearance was achieved, even in the one-column process. Furthermore, we designed an innovative column-free flow-through purification process based on acid precipitation, clarification, ultrafiltration/dilution, and the implementation of an AC filter membrane and an anion exchange chromatography membrane. With this process in the pilot-scale, HCP level can be reduced to below 10 ng/mg, and HMW and LMW can be removed to below 1% while improving mAb recovery. From these results, it is strongly expected that AC is a promising candidate for the next generation of mAb purification processes to improve the economy and efficiency of the process.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Carvão Vegetal/química , Cromatografia por Troca Iônica/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus
6.
Talanta ; 199: 472-477, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952286

RESUMO

Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Durapatita/química , Muramidase/isolamento & purificação , Nanopartículas/química , Soroalbumina Bovina/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Bovinos , Muramidase/química , Muramidase/metabolismo , Tamanho da Partícula , Pressão , Soroalbumina Bovina/química , Propriedades de Superfície , Fatores de Tempo
7.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
8.
J Chromatogr A ; 1598: 101-112, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30954243

RESUMO

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia em Gel , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peso Molecular
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 174-179, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975284

RESUMO

Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin (TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1:1600 and 1:6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×105. The half inhibitory concentration (IC50) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×109 L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.


Assuntos
Anticorpos Monoclonais , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Reações Cruzadas , Diterpenos/metabolismo , Ensaio de Imunoadsorção Enzimática , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
10.
J Chromatogr A ; 1595: 28-38, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898377

RESUMO

The repertoire of complex proteins produced by the host cell during monoclonal antibody (mAb) production has generated a bottleneck in downstream bioprocessing. Low ppm levels of host cell proteins (HCPs) must be achieved at the downstream purification process stage to generate an end product suitable for use in humans. The increased demand for mAb drug products globally has driven research to focus on affordability of mAb production platforms. This has fuelled advancements in manufacturing R&D to deliver higher product titres with better economics without sacrificing product quality. This study highlights the beneficial effects of inclusion of the Emphaze™ AEX Hybrid Purifier, compared to a conventional clarification process, for removal problematic HCPs during downstream bioprocessing of mAbs. Advanced proteomic methods were used to track and identify known 'problematic' HCPs through a multi-cycle Protein A purification process. Removal of histone proteins was observed, along with an average total HCP reduction of 38-fold and an average reduction of 2.3 log in HCDNA concentration. Chromatographic clarification using the Emphaze™ AEX Hybrid Purifier in conjunction with Protein A chromatography resulted in the removal of problematic HCPs including 78 kD glucose-regulated protein, nidogen-1, heat shock proteins, actin, serine protease HTRA1 and matrix metalloproteinase-19. It is shown herein that the Emphaze™ AEX Hybrid Purifier, which is readily incorporated into a mAb purification process during the clarification stage, has the potential to increase Protein A resin lifetime and potentially reduce the number of subsequent polishing chromatographic steps needed to remove HCPs that have a tendency to co-purify with mAb products.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia/métodos , Animais , Humanos , Proteína Estafilocócica A/química
11.
Anal Bioanal Chem ; 411(11): 2425-2437, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30880351

RESUMO

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances. Graphical abstract.


Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Imunoglobulina G/análise , Anticorpos Monoclonais/isolamento & purificação , Simulação por Computador , Contaminação de Medicamentos , Imunoglobulina G/isolamento & purificação , Modelos Químicos , Dodecilsulfato de Sódio/química
12.
J Chromatogr A ; 1597: 100-108, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30922716

RESUMO

Platform manufacturing processes are widely adopted to simplify and standardize the development and manufacturing of monoclonal antibodies (mAbs). However, there are mAbs that do not conform to a platform design due to instability or other protein properties leading to a negative impact on product quality or process performance (non-platform mAb). Non-platform mAbs typically require prolonged development times and significant deviations from the platform process to address these issues due to the need to sequentially optimize individual process steps. In this study, we describe an IgG2 mAb (mAb A) that is susceptible to aggregation and reversible self-association (RSA) under platform conditions. In lieu of a sequential optimization approach, we evaluated the solution stability of mAb A across the platform operating space (solution stability screen). This screening design was used to identify interacting parameters that affected the non-platform mAb stability. A subsequent response surface design was found to predict an acceptable operating space that minimized aggregate formation and RSA across the entire process. This information guided the selection of optimal parameters best suited to avoid destabilizing conditions for each process step. Substantial time savings was achieved by focusing development around these factors including protein concentration, buffer pH, salt concentration, and excipient type. In addition, this work enabled the optimization of a cation exchange chromatography step that removed aggregate without yield losses due to the presence of reversible aggregation. The final optimized process derived from this study resulted in an increase in yield of ˜30% over the original process while maintaining the same level of aggregate clearance to match product quality. Solution stability screening is readily adapted to high throughput technologies to minimize material requirements and accelerate analytical data availability. Implementation of high throughput approaches will further expedite process development and enable enhanced selection of candidate drugs by including process development objectives.


Assuntos
Anticorpos Monoclonais/química , Biotecnologia/métodos , Cromatografia , Anticorpos Monoclonais/isolamento & purificação , Biotecnologia/instrumentação , Cátions/química , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Cloreto de Sódio
13.
Biosens Bioelectron ; 131: 280-286, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30849728

RESUMO

In the study, we describe an oscillatory flow-assisted efficient target enrichment method by using a particle-based microarray device. Periodic oscillating flow effectively increased the mixing and binding performance between the target molecules in the sample solution and surface functionalized microparticles. Particles were trapped, secured, and released with an elastic microvalve structure operated via differences in the flow conditions. Single particle (20-µm diameter) trapping efficiency exceeded 95%. Secured particles can freely move inside each array element based on oscillating sample flow. Furthermore, the particles can be released from the array and collected at the outlet of the device, and this provides an opportunity for further off-chip analysis. As a proof-of-concept, we used the interaction between streptavidin-coated microparticles and fluorescence labeled biotin solution and demonstrated that target enrichment and detection based on oscillatory flow were significantly more efficient than that based on unidirectional or static flow. The applicability of the method was further examined by conducting an on-chip immunoassay to detect the presence of anti-Zika nonstructural protein 1 (NS1) monoclonal antibody. The limit of detection (LOD) was as low as 1 ng/mL with an assay time of only 10 min and less than 10 µL of sample consumption.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Técnicas Biossensoriais , Proteínas não Estruturais Virais/isolamento & purificação , Infecção por Zika virus/diagnóstico , Anticorpos Monoclonais/imunologia , Humanos , Imunoensaio , Limite de Detecção , Técnicas Analíticas Microfluídicas , Tamanho da Partícula , Soluções/química , Propriedades de Superfície , Proteínas não Estruturais Virais/imunologia , Infecção por Zika virus/virologia
14.
Res Vet Sci ; 124: 178-185, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904721

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Vírus da Febre Aftosa/imunologia , Vacinação/veterinária , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Western Blotting , Linhagem Celular , Sequência Conservada , Mapeamento de Epitopos , Epitopos/genética , Feminino , Vírus da Febre Aftosa/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas não Estruturais Virais/genética
15.
Mol Med Rep ; 19(5): 3889-3895, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896845

RESUMO

The carcinoembryonic antigen, glypican­3 (GPC3), is a putative therapeutic target and diagnostic marker of hepatoma. In the present study, a monoclonal antibody (mAb) specifically against GPC3 was obtained via cloning the sequence of GPC3 via polymerase chain reaction and inserting it into a pET16b vector prior to transfection into Escherichia coli (E. coli) BL21. BALB/c mice were immunized with 20 µg purified antigen by intrasplenic embedding. Splenocytes and mouse myeloma cells SP2/0 were fused; then, the hybridoma cells were screened by an indirect ELISA. The properties of the mAb were examined by western blotting and immunofluorescence analysis against the purified protein. The results revealed that the prokaryotic expression vector of GPC3 had been successfully generated and GPC3 was stably expressed in E. coli BL21. A stable hybridoma cell line, 2F3, was generated in the present study, which produced mAbs against GPC3. The mAb 2F3 had a high antibody titer and the isotype was identified as IgG1/κ; 2F3 hybridomas had a median chromosome number of 98. Western blot and immunofluorescence analyses revealed that 2F3 specifically recognized recombinant and native GPC3. The 2F3 clone was proposed as a stable secretor of this mAb against GPC3. The results of present study indicated that the successful preparation of recombinant GPC3 protein and an anti­human GPC3 mouse mAb may be provide a basis for developments in the diagnosis and treatment of liver cancer.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Carcinoma Hepatocelular/metabolismo , Glipicanas/imunologia , Hibridomas/imunologia , Neoplasias Hepáticas/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Carcinoma Hepatocelular/imunologia , Feminino , Glipicanas/genética , Glipicanas/metabolismo , Humanos , Hibridomas/metabolismo , Imunização , Neoplasias Hepáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas
16.
Nat Microbiol ; 4(6): 1024-1034, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30886361

RESUMO

Influenza viruses possess two surface glycoproteins, haemagglutinin and neuraminidase (NA). Although haemagglutinin plays a major role as a protective antigen, immunity to NA also contributes to protection. The NA protein consists of a stalk and a head portion, the latter of which possesses enzymatic NA (or sialidase) activity. Like haemagglutinin, NA is under immune pressure, which leads to amino acid alterations and antigenic drift. Amino acid changes accumulate around the enzymatic active site, which is located at the top of the NA head. However, amino acid alterations also accumulate at the lateral surface of the NA head. The reason for this accumulation remains unknown. Here, we isolated seven anti-NA monoclonal antibodies (mAbs) from individuals infected with A(H1N1)pdm09 virus. We found that amino acid mutations on the lateral surface of the NA head abolished the binding of all of these mAbs. All seven mAbs activated Fcγ receptor (FcγR)-mediated signalling pathways in effector cells and five mAbs possessed NA inhibition activity, but the other two did not; however, all seven protected mice from lethal challenge infection through their NA inhibition activity and/or FcγR-mediated antiviral activity. Serological analysis of individuals infected with A(H1N1)pdm09 virus revealed that some possessed or acquired the anti-NA-lateral-surface antibodies following infection. We also found antigenic drift on the lateral surface of the NA head of isolates from 2009 and 2015. Our results demonstrate that anti-lateral-surface mAbs without NA inhibition activity can provide protection by activating FcγR-mediated antiviral activity and can drive antigenic drift at the lateral surface of the NA head. These findings have implications for NA antigenic characterization in that they demonstrate that traditional NA inhibition assays are inadequate to fully characterize NA antigenicity.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Influenza A/enzimologia , Vírus da Influenza A/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Antígenos Virais/genética , Antivirais/farmacologia , Modelos Animais de Doenças , Entropia , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Evasão da Resposta Imune , Imunidade , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Receptores de IgG , Análise de Sequência de Proteína , Proteínas Virais/genética
17.
MBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782664

RESUMO

Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.


Assuntos
Antivirais/farmacologia , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antivirais/síntese química , Linhagem Celular , Cercopithecus aethiops , Humanos , Ligação Proteica , Conformação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Ensaio de Placa Viral
18.
J Sep Sci ; 42(9): 1816-1827, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30811843

RESUMO

Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida/métodos , Animais , Anticorpos Monoclonais/química , Cromatografia Líquida/tendências , Humanos , Proteína Estafilocócica A/química
19.
MAbs ; 11(3): 569-582, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30668249

RESUMO

Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.


Assuntos
Anticorpos Monoclonais , Polissacarídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Glicosilação , Humanos , Espectrometria de Massas/métodos , Polissacarídeos/química , Polissacarídeos/isolamento & purificação
20.
Cell Host Microbe ; 25(1): 49-58.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629918

RESUMO

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Ebolavirus/patogenicidade , Furões/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Linhagem Celular , Cercopithecus aethiops , Modelos Animais de Doenças , Feminino , Filoviridae/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Infecções por Filoviridae/virologia , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Células Matadoras Naturais , Macaca , Macaca fascicularis , Masculino , Primatas , Análise de Sobrevida , Resultado do Tratamento , Proteínas Virais/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA