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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
2.
Int J Mol Sci ; 21(18)2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32911608

RESUMO

Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a 'plug and play' manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes-the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins-and elaborate on the benefits as well as drawbacks of the different technologies.


Assuntos
Anticorpos Biespecíficos/análise , Anticorpos Biespecíficos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Animais , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos
3.
MAbs ; 12(1): 1731938, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32151188

RESUMO

Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/análise , Ensaios de Triagem em Larga Escala/métodos , Inteínas , Animais , Humanos , Engenharia de Proteínas/métodos
4.
Bioresour Technol ; 129: 421-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23262020

RESUMO

Acetone, butanol and ethanol were produced in a continuous two-stage fermentation integrated with pervaporation using freely suspended cells of C. acetobutylicum ATCC 824. PDMS composite pervaporation membranes were directly coupled to the second fermentor which lead to decreased solvent titers. Overall productivity was increased from 0.45 g L(-1) h(-1) to 0.88 g L(-1) h(-1) when increasing the carbohydrate concentration in the feed from 60 to 120 g L(-1). The highest overall productivity of 1.13 g L(-1) h(-1) was achieved when increasing the carbohydrate concentration further to 150 g L(-1) even though productivity decreased significantly in the first fermentor due to substrate inhibition. In this phase that lasted 200 h, the average flux reached 0.621 kg m(-2) h(-1) and the total solvent concentration in the permeate was 202 g L(-1). High solvent titers in the second fermentor were beneficial for the performance of the pervaporation unit leading to higher fluxes and total solvent concentrations in the permeate.


Assuntos
Acetona/metabolismo , Reatores Biológicos/microbiologia , Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Etanol/metabolismo , Membranas Artificiais , Desenho de Equipamento , Análise de Falha de Equipamento , Gases/química , Porosidade , Integração de Sistemas
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