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1.
J Biosci Bioeng ; 120(3): 340-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25683450

RESUMO

To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.


Assuntos
Anticorpos Monoclonais/biossíntese , Separação Celular/métodos , Citometria de Fluxo/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/genética , Técnicas de Cultura Celular por Lotes , Células CHO , Tamanho Celular , Cricetinae , Cricetulus , Fluoresceína-5-Isotiocianato , Vetores Genéticos/genética , Humanos , Transfecção
2.
Appl Microbiol Biotechnol ; 97(6): 2531-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22926643

RESUMO

Secretory capacities including folding and assembly are believed to be limiting factors in the establishment of mammalian cell lines producing high levels of recombinant therapeutic proteins. To achieve industrial success, it is also important to improve protein folding, assembly, and secretory processes in combination with increasing transcription and translation. Here, we identified the expression of CHOP/Gadd153 and GRP78, which are unfolded protein response (UPR)-related genes, correlated with recombinant antibody production in stable CHO cells. Subsequently, CHOP overexpression resulted in increasing recombinant antibody production in some mammalian cell lines, and in addition a threefold further enhancement was obtained by combining expression with UPR-related genes or ER chaperones in transient assays. Overexpression of CHOP had no effect on the biochemical characteristics of the product. These results suggest overexpression of CHOP and its combinations may be an effective method to efficiently select a single cell line with a high level of antibody production in the development of cell lines for manufacturing.


Assuntos
Anticorpos/metabolismo , Biotecnologia/métodos , Proteínas de Choque Térmico/biossíntese , Fator de Transcrição CHOP/biossíntese , Animais , Anticorpos/genética , Células CHO , Cricetinae , Cricetulus , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição CHOP/genética
3.
Biol Pharm Bull ; 25(12): 1537-45, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12499636

RESUMO

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. We have previously obtained the mouse anti-Fas antibody HFE7A (m-HFE7A), which specifically induces apoptosis in inflammatory cells. In order to apply m-HFE7A for human therapy, we performed antibody humanization of m-HFE7A by grafting the mouse complementarity-determining regions (CDRs) to a human antibody. Five versions of humanized HFE7A (h-HFE7A) demonstrated the same antigen-binding affinity and same competition-binding activity against Fas as the chimeric HFE7A. Furthermore, these h-HFE7As induced the same degree of apoptosis in WR19L12a cells that express human Fas on their surface as chimeric HFE7A does. To further probe the structural basis for antibody humanization, we determined the three-dimensional structure of the h-HFE7A antigen-binding fragment (Fab) by X-ray crystallography and compared it with the crystal structure of the parent m-HFE7A Fab previously determined. The main-chain conformation in each h-HFE7A CDR is almost identical to that in m-HFE7A with root mean square (rms) deviations of 0.14-0.77 A. However, a significant segmental shift was observed in the CDR-L1 loop. Together with the high temperature factors of the CDR-L1 residues, both the loops are flexible, suggesting that the CDR-L1 loop would undergo conformational change upon binding to the antigen. Our results indicate that the humanization of m-HFE7A succeeded in maintaining the main-chain conformation as well as the flexibility of the CDR loop.


Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Apoptose/imunologia , Sítios de Ligação de Anticorpos , Ligação Competitiva/imunologia , Células COS , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Cristalização , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Dados de Sequência Molecular
4.
Protein Pept Lett ; 9(3): 259-63, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12144523

RESUMO

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.


Assuntos
Anticorpos Monoclonais/química , Fragmentos de Imunoglobulinas/química , Receptor fas/química , Animais , Anticorpos Monoclonais Murinos , Cristalografia por Raios X , Humanos , Camundongos
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