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51.
Yolande Rouiller Arnaud Périlleux Natacha Collet Martin Jordan Matthieu Stettler Hervé Broly 《MABS-AUSTIN》2013,5(3):501-511
An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame. 相似文献
52.
Marcus Weinguny Peter Eisenhut Gerald Klanert Nikolaus Virgolini Nicolas Marx Andreas Jonsson Daniel Ivansson Ann Lövgren Nicole Borth 《Biotechnology and bioengineering》2020,117(11):3435-3447
Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones. 相似文献
53.
Online control of cell culture redox potential prevents antibody interchain disulfide bond reduction
Michael W. Handlogten Jihong Wang Sanjeev Ahuja 《Biotechnology and bioengineering》2020,117(5):1329-1336
The phenomenon of monoclonal antibody (mAb) interchain disulfide bond reduction during manufacturing processes continues to be a focus of the biotechnology industry due to the potential for loss of product, increased complexity of purification processes, and reduced stability of the drug product. We hypothesized that antibody reduction can be mitigated by controlling the cell culture redox potential and subsequently established a threshold redox potential above which the mAb remained intact and below which there were significant and highly variable amounts of reduced mAb. Using this knowledge, we developed three control schemes to prevent mAb reduction in the bioreactor by controlling the cell culture redox potential via an online redox probe. These control methodologies functioned by increasing the concentration of dissolved oxygen (DO), copper (II) (Cu), or both DO and Cu to maintain the redox potential above the threshold value. Using these methods, we were able to demonstrate successful control of antibody reduction. Importantly, the redox control strategies did not significantly impact the cell growth, viability, mAb production, or product quality attributes including aggregates, C-terminal lysine, high mannose, deamidation, and glycation. Our results demonstrate that controlling the cell culture redox potential is a simple and effective method to prevent mAb reduction. 相似文献
54.
Natascha Verhagen Attila Teleki Christoph Heinrich Martin Schilling Andreas Unsöld Ralf Takors 《Biotechnology and bioengineering》2020,117(11):3239-3247
The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5′-adenosyl)- l -methionine (SAM) and 5′-deoxy-5′-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells. 相似文献
55.
56.
Neuropilins (Nrp) are type I transmembrane proteins that function as receptors for vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3) ligand families. Sema3s function as potent endogenous angiogenesis inhibitors but require proteolytically processing by furin to compete with VEGF for Nrp binding. This processing liberates a C-terminal arginine (CR) that is necessary for binding to the b1 domain of Nrp, a common feature shared by Nrp ligands. The CR is necessary but not sufficient for potent Nrp inhibition, and the role of upstream residues is unknown. We demonstrate that the second-to-last residue (C-1), immediately upstream of the CR, plays a significant role in controlling competitive ligand binding by orienting the C-terminus for productive Nrp binding. With the use of a peptide library derived from Sema3F, C-1 residues that preferentially adopt an extended bound-like conformation, including proline and β-branched amino acids, were found to produce the most avid competitors. Consistent with this, analysis of the binding thermodynamics revealed that more favorable entropy is responsible for the observed binding enhancement of C-1 proline. We further tested the effect of the C-1 residue on Sema3F processing by furin and found an inverse relationship between processing and inhibitory potency. Analysis of all Sema3 family members reveals two non-equivalent furin processing sites differentiated by the presence of either a C-1 proline or a C-1 arginine and resulting in up to a 40-fold difference in potency. These data reveal a novel regulatory mechanism of Sema3 activity and define a fundamental mechanism for preferential Nrp binding. 相似文献
57.
Marian L. Kruzel Jeffrey K. Actor Michał Zimecki Jasen Wise Paulina Płoszaj Shaper Mirza Mark Kruzel Shen-An Hwang Xueqing Ba Istvan Boldogh 《Journal of biotechnology》2013
Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. 相似文献
58.
Lianchun Fan Ibrahim KaduraLara E. Krebs Jeffery L. LarsonDaniel M. Bowden Christopher C. Frye 《Journal of biotechnology》2013
Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed. 相似文献
59.
Gargi Seth Robert W. Hamilton Thomas R. Stapp Lisa Zheng Angela Meier Krista Petty Stephenie Leung Srikanth Chary 《Biotechnology and bioengineering》2013,110(5):1376-1385
Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 106 cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385. © 2012 Wiley Periodicals, Inc. 相似文献
60.
Kyoung Ho Lee Tomomi Tsutsui Kohsuke Honda Hisao Ohtake Takeshi Omasa 《Cytotechnology》2013,65(6):1017-1026
The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression. 相似文献