Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Bioorg Chem ; 112: 104863, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823405

RESUMO

The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.


Assuntos
Antibacterianos/metabolismo , Desenvolvimento de Medicamentos , Glicosídeo Hidrolases/metabolismo , Trastuzumab/metabolismo , alfa-L-Fucosidase/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Biotinilação , Relação Dose-Resposta a Droga , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/genética , Fenômenos Magnéticos , Estrutura Molecular , Mutação , Relação Estrutura-Atividade , Trastuzumab/química , Trastuzumab/isolamento & purificação , alfa-L-Fucosidase/genética
2.
Nat Biomed Eng ; 4(11): 1044-1052, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32690883

RESUMO

Efficient purification is crucial to providing large quantities of recombinant therapeutic proteins, such as monoclonal antibodies and cytokines. However, affinity techniques for manufacturing protein therapeutics that use biomolecule-conjugated agarose beads that harness specific biomolecular interactions suffer from issues related to protein denaturation, contamination and the need to maintain biomolecule-specific conditions for efficient protein capture. Here, we report a versatile and scalable method for the purification of recombinant protein therapeutics. The method exploits the high-affinity and controllable host-guest interactions between cucurbit[7]uril (CB[7]) and selected guests such as adamantylammonium. We show that the Herceptin (the brand name of trastuzumab, a monoclonal antibody drug used to treat breast cancer) and the much smaller cytokine interferon α-2a can be purified by site-specifically tagging them with adamantylammonium using the enzyme sortase A, followed by high-affinity binding with CB[7]-conjugated agarose beads and the recovery of the protein using a guest with a stronger affinity for CB[7]. The thermal and chemical stability of CB[7] beads and their scalability, recyclability and low cost may also make them advantageous for the manufacturing of biosimilars.


Assuntos
Cromatografia em Agarose/métodos , Interferon alfa-2/química , Interferon alfa-2/isolamento & purificação , Trastuzumab/química , Trastuzumab/isolamento & purificação , Hidrocarbonetos Aromáticos com Pontes/química , Humanos , Imidazóis/química
3.
Analyst ; 145(8): 3148-3156, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32191233

RESUMO

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng µL-1 compared to 0.05 ng µL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.


Assuntos
Cromatografia de Afinidade/métodos , Enzimas Imobilizadas/química , Membranas Artificiais , Trastuzumab/análise , Sequência de Aminoácidos , Animais , Masculino , Estudo de Prova de Conceito , Proteólise , Ratos Sprague-Dawley , Proteína Estafilocócica A/química , Fatores de Tempo , Trastuzumab/química , Trastuzumab/isolamento & purificação , Trastuzumab/farmacocinética , Tripsina/química
4.
Genes (Basel) ; 12(1)2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33396657

RESUMO

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.


Assuntos
Anticorpos Monoclonais/biossíntese , Sistemas CRISPR-Cas , Galinhas/genética , Clara de Ovo/química , Receptor ErbB-2/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Feminino , Edição de Genes/métodos , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Trastuzumab/biossíntese , Trastuzumab/isolamento & purificação , Zigoto/química , Zigoto/metabolismo
5.
Sci Rep ; 9(1): 671, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679500

RESUMO

IgG is an indispensable biological experimental tool as well as a widely-used therapeutic protein. However, cell culture-based expression of monoclonal IgG is costly and time-consuming, making this process difficult to use for high-throughput screening in early-stage evaluation of biologics. With the goal of establishing a fast, simple, and robust high-throughput expression system for IgG, we implemented the synthesis of functional aglycosylated IgG by constructive approach based on a reconstituted prokaryotic cell-free protein synthesis system (PURE system). Optimization of the PURE system revealed that the following factors and reaction conditions were needed for IgG synthesis: (1) inclusion of the disulfide bond isomerase DsbC, (2) adjustment of the GSH/GSSG ratio, (3) inclusion of the molecular chaperone DnaK and its cofactors, and (4) use of an extended incubation time. Synthesis temperature and template DNA ratio (light chain-/heavy chain-encoding) also had been optimized for each IgG. Under optimal conditions, peak production of the anti-HER2 antibody trastuzumab reached 124 µg/mL. Furthermore, the active forms of other IgGs, including IgG1, IgG2, and IgG4 subclasses, also were synthesized. These results provide basic information for the development of novel high-throughput expression and functional screening systems for IgG, as well as useful information for understanding the IgG synthesis process.


Assuntos
Imunoglobulina G/biossíntese , Engenharia de Proteínas/métodos , Linhagem Celular , Sistema Livre de Células , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutationa/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Oxirredução , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptor ErbB-2/imunologia , Solubilidade , Trastuzumab/biossíntese , Trastuzumab/isolamento & purificação , Trastuzumab/farmacologia
6.
J Nucl Med ; 60(3): 418-423, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30630938

RESUMO

Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use.


Assuntos
Cetuximab/química , Imagem Óptica/métodos , Pesquisa Translacional Biomédica , Trastuzumab/química , Cetuximab/isolamento & purificação , Descoberta de Drogas , Controle de Qualidade , Trastuzumab/isolamento & purificação
7.
Eur J Mass Spectrom (Chichester) ; 25(3): 324-332, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30351978

RESUMO

Capillary electrophoresis-mass spectrometry coupling is a growing technique in biopharmaceutics characterization. Assessment of monoclonal antibodies is well known at middle-up and bottom-up levels to obtain information about the sequence, post-translational modifications and degradation products. Intact protein analysis is an actual challenge to be closer to the real protein structure. At this level, actual techniques are time consuming or cumbersome processes. In this work, a 20 minutes separation method has been developed to optimize characterization of intact monoclonal antibodies. Thus, separation has been done on a positively charged coated capillary with optimized volatile background electrolyte and sample buffer. Three world-wide health authorities approved monoclonal antibodies have been used to set up a rapid and ease of use method. Intact trastuzumab, rituximab and palivizumab isoforms have been partially separated with this method in less than 20 minutes under denaturing conditions. For each monoclonal antibody, 2X-glycosylated and 1X-glycosylated structures have been identified and separated. Concerning basic and acidic variants, potential aspartic acid isomerization modification and asparagine deamidation have been observed. Accurate mass determination for high-mass molecular species remains a challenge, but the progress in intact monoclonal antibodies separation appears very promising for biopharmaceutics characterization.


Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Trastuzumab/química , Glicosilação , Isomerismo , Processamento de Proteína Pós-Traducional , Trastuzumab/isolamento & purificação
8.
Anal Chem ; 90(20): 12161-12167, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30207156

RESUMO

Rapid, convenient methods for monoclonal antibody (mAb) isolation are critical for determining the concentrations of therapeutic mAbs in human serum. This work uses porous nylon membranes modified with a HER2 peptide mimotope, KGSGSGSQLGPYELWELSH (KH19), for rapid affinity capture of Herceptin, a mAb used to treat breast cancer. Covalent linking of KH19 to poly(acrylic acid)-containing films in porous nylon leads to a Herceptin-binding capacity of 10 mg per mL of membrane and allows selective Herceptin capture from diluted (1:3) human serum in 5 min. Liquid chromatography-mass spectrometry demonstrates the high purity of eluted Herceptin. Moreover, the fluorescence intensity of the protein eluted from membranes increases linearly with the amount of Herceptin spiked in loading solutions containing diluted (1:3) human serum. These results demonstrate the promise of mimotope-modified membranes for Herceptin analysis that does not require secondary antibodies or derivatization with fluorescent labels. Thus, mimotope-containing membranes may form part of a simple benchtop analysis system for assessing the concentrations of therapeutic mAbs.


Assuntos
Proteínas Imobilizadas/química , Fragmentos de Peptídeos/química , Receptor ErbB-2/química , Trastuzumab/análise , Trastuzumab/isolamento & purificação , Adsorção , Humanos , Nylons/química , Tamanho da Partícula , Porosidade , Propriedades de Superfície , Trastuzumab/sangue
9.
Methods Mol Biol ; 1827: 367-380, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196507

RESUMO

Core fucosylation plays a critical role in modulating the effector functions of therapeutic antibodies such as the antibody-dependent cellular cytotoxicity (ADCC) through adversely affecting the affinity of antibodies for Fcγ receptors. Thus, a facile method for Fc defucosylation of antibodies is important both for functional studies and for an enhanced therapeutic efficacy. In this chapter, we describe a detailed protocol for chemoenzymatic defucosylation of antibodies using Herceptin (trastuzumab) as a model system. The protocol includes (a) Fc deglycosylation using endoglycosidase S2 (Endo-S2); (b) enzymatic defucosylation of the resulting Fucα1,6GlcNAc-Herceptin using two distinct bacterial α-fucosidases, AlfC and BfFuc; (c) transglycosylation of the GlcNAc-Herceptin using an Endo-S2 mutant (Endo-S2 D184M) as the enzyme and a complex N-glycan oxazoline as the donor substrate; and (d) SPR analysis of the binding of antibody glycoforms with the FcγIIIA receptor. The protocol of enzymatic defucosylation of Herceptin should be equally applicable for the Fc glycan engineering of other mAbs.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Bactérias/enzimologia , Engenharia de Proteínas/métodos , alfa-L-Fucosidase/metabolismo , Cromatografia Líquida , Glicosilação , Humanos , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Trastuzumab/isolamento & purificação
10.
Bioanalysis ; 10(11): 851-862, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29863890

RESUMO

AIM: Compared with small molecules, LC-MS quantitation of larger biotherapeutic proteins such as antibodies and antibody-drug conjugates at the intact level presents many challenges in both LC and MS due to their higher molecular weight, bigger size, structural complexity and heterogeneity. RESULTS & CONCLUSION: In this study, quantitation of an intact lysine-linked antibody-drug conjugate, trastuzumab emtansine is presented. Trastuzumab emtansine was extracted from rat plasma using bead-based immunoaffinity capture; after elution from the beads, it was directly analyzed on a LC-HRMS system. Quantitation using both extracted ion chromatogram and deconvoluted mass peaks was evaluated. A limit of quantitation was approximately 20 ng on column with a linear dynamic range from 5 to 100 µg/ml. In addition, the reproducibility and distribution of the drug-to-antibody ratio at different trastuzumab emtansine concentrations were discussed.


Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida/métodos , Imunoconjugados/sangue , Espectrometria de Massas/métodos , Maitansina/análogos & derivados , Trastuzumab/sangue , Ado-Trastuzumab Emtansina , Animais , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Limite de Detecção , Maitansina/sangue , Maitansina/química , Maitansina/isolamento & purificação , Ratos , Trastuzumab/química , Trastuzumab/isolamento & purificação
11.
Protein Expr Purif ; 150: 109-118, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29857036

RESUMO

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems.


Assuntos
Brevibacillus/metabolismo , Expressão Gênica , Fragmentos Fab das Imunoglobulinas , Trastuzumab , Brevibacillus/genética , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Trastuzumab/biossíntese , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/isolamento & purificação
12.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1055-1056: 158-164, 2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28477519

RESUMO

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3-5µm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.


Assuntos
Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Trastuzumab/isolamento & purificação , Alemtuzumab , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Células CHO , Resinas de Troca de Cátion/química , Cricetinae , Cricetulus , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos , Trastuzumab/química
13.
J Vis Exp ; (119)2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28190027

RESUMO

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.


Assuntos
Anticorpos/isolamento & purificação , Cromatografia de Afinidade/métodos , Vetores Genéticos , Transfecção/métodos , Anticorpos/genética , Técnicas de Cultura Celular por Lotes/métodos , Contagem de Células , Cromatografia de Afinidade/instrumentação , Células HEK293 , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Polietilenoimina , Trastuzumab/isolamento & purificação
14.
Methods Mol Biol ; 1466: 179-84, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27473490

RESUMO

Monoclonal antibodies (mAbs) are widely used in cancer therapy and recently many new mAbs have gained EMA and FDA approvals for oncology indications. Here we describe a highly reproducible CZE method, relying on a cationic coating allowing separation and identification of a complex mixture of four compounded mAbs widely used in cancer therapy (cetuximab, rituximab, bevacizumab, and trastuzumab).


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antineoplásicos Imunológicos/isolamento & purificação , Eletroforese Capilar/métodos , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Bevacizumab/administração & dosagem , Bevacizumab/isolamento & purificação , Cetuximab/administração & dosagem , Cetuximab/isolamento & purificação , Composição de Medicamentos , Humanos , Controle de Qualidade , Rituximab/administração & dosagem , Rituximab/isolamento & purificação , Trastuzumab/administração & dosagem , Trastuzumab/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...