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1.
Anal Bioanal Chem ; 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32030491

RESUMO

Immunoglobin E (IgE)-related allergy constitutes a high proportion in allergic diseases. The production of specific IgE is key to evoking serial cascades and pathological reactions. Thus, targeting IgE is a different therapeutic approach from symptomatic treatments. Monoclonal antibodies (mAbs) against IgE were developed and a humanized antibody, omalizumab, was approved by five countries. It could inhibit the binding of IgE with epsilon receptor I of crystallizable fragment (FcεRI), thus preventing anaphylactic reactions. However, no bioactivity assay, which is the critical quality attribute and should thoroughly reflect the clinical mechanism, has been established to date. In commercial lot release, only the enzyme-linked immunosorbent assay (ELISA) method was applied, which only reflects the binding of omalizumab to IgE but not the subsequent reaction. In scientific research works, human FcεRI-transfected RBL-2H3 cells were used to indicate degranulation based on the detection of ß-hexosaminidase. Nevertheless, this method needs much work to stabilize the response and, hence, is not suitable for routine usage in commercial production and control of antibodies. To evaluate the bioactivity of anti-IgE antibodies including omalizumab using a simple assay that reflects the following mechanism of actions (MOA) after binding, we established an RBL-2H3 cell line transfected with both the α subunit of human FcεRI and nuclear factor-activated T cell (NFAT) response elements, the latter is conjugated with a luciferase gene, which could shed luminescence when substrates exist. The method was proven to possess good specificity, accuracy, linearity, and precision and may be utilized as a supplement to anti-IgE antibody bioactivity assays in terms of development, lot release, stability, and comparability studies. Graphical abstract The mechanism sketch of reporter gene assay for bioactivity determination of anti-IgE antibodies by RBL-2H3/FcεRIα/NFAT-Luc cells (left) and representative curves generated by the reporter gene assay (right).

2.
Anal Chem ; 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31983199

RESUMO

BVZ-BC (bevacizumab-biosimilar candidate) is a proposed biosimilar to bevacizumab. Bevacizumab binds to vascular endothelial growth factor (VEGF) type A and prevents the interaction of VEGF with its receptors on the surface of endothelial cells, neutralizing angiogenesis required for the growth, persistence, and metastases of solid tumors. An analytical comparison of BVZ-BC and bevacizumab was performed using state-of-the-art analytical techniques, including biochemical and biophysical characterization, biological activity, and immunological properties. Multiple attributes of the molecules were evaluated, including amino acid sequence, disulfide structure, glycan profiles, free thiol content, isoelectric point, protein content, subvisible particles, higher-order structure such as near- and far-ultraviolet circular dichroism and differential scanning calorimetry, product purity and product-related impurities, and process-related impurities. Biological activity assessment employed orthogonal assays such as the VEGF cell-based bioassay and the VEGF enzyme-linked immunosorbent assay, and Fc functional assays to interrogate all expected biological activities. An accumulation of 18 batches of bevacizumab (sourced in China, manufactured in Europe, Roche) and 10 batches of BVZ-BC representing unique drug product lots from each individual drug substance lot were utilized in this study. The analytical similarity between BVZ-BC and bevacizumab was assessed and demonstrated the similarities of all of the quality attributes between BVZ-BC and bevacizumab.

3.
Emerg Microbes Infect ; 8(1): 1584-1592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682199

RESUMO

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.

4.
Jpn J Infect Dis ; 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31666494

RESUMO

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signalling molecule that causes the excessive secretion of various proinflammatory factors induced by pyrogens. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. The RGA could detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 µg/ml, and 1µg/ml, respectively. The method had good precision and accuracy and could be applied to many products [e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, Haemophilus influenza type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, group A and group C meningococcal conjugate vaccine, diphtheria, tetanus, pertussis (acellular, component), poliomyelitis (inactivated) vaccine, and imject alum adjuvant]. The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.

5.
Biotechnol Adv ; : 107466, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31697994

RESUMO

Complex structure and structure-function relationship of biopharmaceuticals require extensive analytical characterization and appropriate quality control of the products. Despite rapid development of sophisticated physicochemical techniques, biological activity measurement remains the critical role in inferring the high-order structure of biopharmaceuticals. Cell-based biological assays are mostly applied to determine the biological activity of biopharmaceuticals, however, refined biological assays are continually needed to increase their robustness. Reporter gene assays (RGAs) which are mechanism of action (MOA) related, less variable, accurate, precise, and labor-saving are becoming more and more recognized and adopted in the quality control. Here we discuss the importance of bioactivity determination, the strength and weakness of various assay formats with RGAs. We also introduce the mechanism of RGAs, and present a number of examples for RGAs to determine the bioactivity of various biopharmaceuticals, which indicate their extensive use in the screening, characterization, quality control, stability and biosimilarity study. We believe that with the rapid development of biotechnology, new strategies of bioassays based on RGAs will be more widely applied in various fields of biopharmaceuticals.

6.
Anal Bioanal Chem ; 410(27): 7067-7075, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30178083

RESUMO

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Avaliação Pré-Clínica de Medicamentos , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/imunologia , Engenharia Celular , Linhagem Celular , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Humanos , Interleucina-6/imunologia , Luciferases/genética , Luciferases/imunologia , Receptores de Interleucina-6/imunologia , Proteínas Recombinantes/imunologia
7.
Electrophoresis ; 39(16): 2091-2098, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29797663

RESUMO

CE is central to the analysis, process development and approval of therapeutic monoclonal antibodies (mAbs). Recently, imaged capillary isoelectric focusing (icIEF) has emerged as a powerful technique for quantitative protein charge heterogeneity monitoring and characterization, particularly for mAbs. However, icIEF has yet to be validated for therapeutically relevant mAbs adhering to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, for the first time, icIEF technology was validated by 10 laboratories across 8 independent companies using a therapeutic mAb. The parameters of this method validation strictly follow the guideline of the ICH. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness. These results represent a significant step forward in standardizing the use of icIEF methods for the clinical approval of therapeutic mAbs.


Assuntos
Anticorpos Monoclonais/análise , Focalização Isoelétrica/métodos , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Guias como Assunto , Focalização Isoelétrica/normas , Laboratórios/normas , Reprodutibilidade dos Testes
8.
Life Sci ; 197: 91-100, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29421438

RESUMO

FUZ is regarded as a planar cell polarity effector that controls multiple cellular processes during vertebrate development. However, the role of FUZ in tumor biology remains poorly studied. Our purpose of this study is to discover the physiological effects and mechanism of FUZ in non-small-cell lung cancer (NSCLC) in vitro. With the help of bioinformatics analysis, we noticed that the expression level of FUZ negatively correlates with prognosis of NSCLC patients. Exogenous FUZ expression markedly promoted cell proliferation of NSCLC cells. The phosphorylation of Erk1/2, STAT3 and related signaling molecules were induced activated after FUZ over-expression. FUZ also plays an important role in cell motility by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). FUZ promotes EMT along with the up-regulation of N-cadherin, vimentin, Zeb1, Twist1 and decreased level of E-cadherin. Furthermore, we also carried out FUZ directed siRNA treatments to prove the above observations. Knockdown of FUZ resulted in delayed cell growth as well as impaired cell migration and reversed EMT phonotype. Importantly, we reported for the first time that FUZ is a BNIP3-interacting protein. Loss of FUZ resulted in decreased BNIP3 protein level, but no influence on BNIP3 mRNA level, suggesting weakened stability of BNIP3 protein. Overall, our results in vitro show that FUZ is responsible for NSCLC progression and metastasis, suggesting that FUZ can be a potential therapeutic target for NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Sistemas de Liberação de Medicamentos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/genética
9.
J Pharm Biomed Anal ; 148: 280-287, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29059618

RESUMO

Eosinophilic asthma is characterized by the eosinophilic inflammation with the allergen independent activation of Th2 lymphocytes. Since IL-5 plays an important role in the maturation, survival and migration of eosinophils, hence the pathogenesis of eosinophilic asthma, biotherapeutics targeting IL-5/IL-5Rα have been developed and/or marketed, including Mepolizumab, Reslizumab, and Benralizumab. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current mode of action (MOA) based method used in the quality control and stability tests for anti-IL-5 mAbs is anti-proliferation assay, which is tedious with long duration and high variation. We describe here the development and validation of a reporter gene assay (RGA), based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements. After careful optimization, we demonstrate the excellent specificity, precision, accuracy and linearity of the established RGA. Our study also proves that the assay is superior on precision, sensitivity and assay simplicity to the anti-proliferation assay. The established RGA is also applicable to another anti-IL-5Rα mAb. These results show for the first time that this novel RGA, based on the IL-5-IL-5R-STAT5 pathway, can be a valuable supplement to the anti-proliferation assay and employed in the bioactivity determination of anti-IL-5/anti-IL-5Rα biotherapeutics.


Assuntos
Anticorpos Monoclonais/farmacologia , Bioensaio/métodos , Genes Reporter/genética , Interleucina-5/metabolismo , Asma/tratamento farmacológico , Asma/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Luciferases/genética , Fator de Transcrição STAT5/metabolismo , Sensibilidade e Especificidade
11.
J Pharm Biomed Anal ; 145: 447-453, 2017 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-28735186

RESUMO

Being regarded as the 'cancer panacea', the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been proved particularly effective in treating various cancers. However, the cell-based bioassay to measure the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here, we describe the development and validation of a reporter gene assay consisting of two-cell systems to measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent performance characteristics including specificity, accuracy and precision. The biological relevance of the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in characterization and development of new anti-PD1/anti-PD-L1 mAbs.


Assuntos
Genes Reporter , Animais , Anticorpos Monoclonais , Antígeno B7-H1 , Células CHO , Cricetulus , Humanos , Neoplasias
12.
MAbs ; 8(7): 1210-1223, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27380163

RESUMO

ASBTRACT The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacologia , Maitansina/análogos & derivados , Trastuzumab/química , Trastuzumab/farmacologia , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Maitansina/química , Maitansina/farmacologia , Mapeamento de Peptídeos , Ressonância de Plasmônio de Superfície
13.
J Pharm Biomed Anal ; 125: 212-8, 2016 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-27042807

RESUMO

Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics.


Assuntos
Bevacizumab/imunologia , Genes Reporter , Fator A de Crescimento do Endotélio Vascular/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Reprodutibilidade dos Testes
14.
Sci Rep ; 7: 20029, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26823113

RESUMO

Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.


Assuntos
Anticorpos Monoclonais/biossíntese , Epitopos/imunologia , Galactose/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sequência de Carboidratos , Humanos , Imunoglobulina G/imunologia , Camundongos , Oligossacarídeos/imunologia , Polissacarídeos/imunologia
15.
Drug Discov Today ; 21(5): 740-65, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26821133

RESUMO

Glycosylation of therapeutic proteins has a profound impact on their safety and efficacy. Many factors shape the glycosylation of biotherapeutics, ranging from expression systems and cell culture processes to downstream purification strategies. Various analytical technologies have been developed to address questions concerning different aspects of glycosylation. Informatics tools are also crucial for a systematic understanding of the glycosylation processes. Hence, an integrated approach is required to harness glycosylation for the production of optimal and consistent glycoprotein-based therapeutic drugs. Here, we review the latest developments and challenges in glycosylation analysis and control in the context of bioprocessing monoclonal antibodies.


Assuntos
Descoberta de Drogas , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Glicoproteínas/metabolismo , Glicosilação , Humanos
16.
Yao Xue Xue Bao ; 50(1): 94-8, 2015 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-25924482

RESUMO

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Genes Reporter , Humanos , Reprodutibilidade dos Testes , Rituximab
17.
Yao Xue Xue Bao ; 49(3): 363-7, 2014 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-24961108

RESUMO

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Imunoconjugados , Preparações Farmacêuticas , Espectrofotometria Ultravioleta/métodos , Anticorpos/análise , Anticorpos/química , Glicosilação , Imunoconjugados/análise , Imunoconjugados/química , Maleimidas/análise , Maleimidas/química , Peso Molecular , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química
18.
Sheng Wu Gong Cheng Xue Bao ; 30(9): 1473-80, 2014 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-25720162

RESUMO

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Mapeamento de Peptídeos , Peptídeos
19.
PLoS One ; 6(11): e26729, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22073188

RESUMO

The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly 300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein. Also of interest are the protease inhibitor, alpha2-macroglobulin (A2m), and the mitochondrial complex II subunit Sdhc, both ageing-related genes found strongly over-expressed in the naked mole-rat. These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat's fascinating characteristics.


Assuntos
Mitocôndrias/genética , Ratos-Toupeira/genética , Análise de Sequência de RNA , Animais , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Molécula de Adesão da Célula Epitelial , Camundongos , Oxirredução , alfa-Macroglobulinas/genética
20.
Cell Signal ; 20(12): 2208-20, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18771726

RESUMO

Cell migration plays a critical role during the development of most organisms and the process of malignant tumor metastasis. In the present study, we investigated the role of PTPIP51 (protein tyrosine phosphatase interacting protein 51) in cell motility. Overexpression of PTPIP51 induced cell elongation, increased cell migration, adhesion, and spreading, while downregulation of PTPIP51 had the opposite effects. We demonstrated here, that PTPIP51 could regulate ERK activity on Raf level, since MEK inhibitor and dominant-negative Raf-1 but not Ras could inhibit the ERK activation induced by PTPIP51. Further studies proved that PTPIP51 could interact with Raf-1 through 14-3-3, suggesting that PTPIP51 is a regulator of the Raf-MEK-ERK cascade through modulation of Raf-1 by 14-3-3. In addition, two redundant 14-3-3 binding domains in the PTPIP51 protein have been identified by deletion/mutation studies. We conclude that PTPIP51 regulates cell morphology and cell motility via interaction with Raf-1 through 14-3-3, and that PTPIP51 binds to 14-3-3 through two redundant binding domains.


Assuntos
Proteínas 14-3-3/metabolismo , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Quinases raf/metabolismo , Sequência de Aminoácidos , Ensaios de Migração Celular , Forma Celular , Citometria de Fluxo , Células HeLa , Humanos , Imunoprecipitação , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno
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