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1.
Biotechnol J ; 15(8): e1900565, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32170810

RESUMO

Chinese hamster ovary (CHO) cells are currently the primary host cell lines used in biotherapeutic manufacturing of monoclonal antibodies (mAbs) and other biopharmaceuticals. Cellular energy metabolism and endoplasmic reticulum (ER) stress are known to greatly impact cell growth, viability, and specific productivity of a biotherapeutic; but the molecular mechanisms are not fully understood. The authors previously employed multi-omics profiling to investigate the impact of a reduction in cysteine (Cys) feed concentration in a fed-batch process and found that disruption of the redox balance led to a substantial decline in cell viability and titer. Here, the multi-omics findings are expanded, and the impact redox imbalance has on ER stress, mitochondrial homeostasis, and lipid metabolism is explored. The reduced Cys feed activates the amino acid response (AAR), increases mitochondrial stress, and initiates gluconeogenesis. Multi-omics analysis reveals that together, ER stress and AAR signaling shift the cellular energy metabolism to rely primarily on anaplerotic reactions, consuming amino acids and producing lactate, to maintain energy generation. Furthermore, the pathways are demonstrated in which this shift in metabolism leads to a substantial decline in specific productivity and altered mAb glycosylation. Through this work, meaningful bioprocess markers and targets for genetic engineering are identified.


Assuntos
Biologia Computacional , Cisteína , Metabolismo Energético , Animais , Células CHO , Biologia Computacional/métodos , Cricetinae , Cricetulus , Cisteína/química , Oxirredução
2.
Biotechnol J ; 14(4): e1800352, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30485675

RESUMO

There is continual demand to maximize CHO cell culture productivity of a biotherapeutic while maintaining product quality. In this study, a comprehensive multi-omics analysis is performed to investigate the cellular response to the level of dosing of the amino acid cysteine (Cys) in the production of a monoclonal antibody (mAb). When Cys feed levels are insufficient, there is a significant decrease in protein titer. Multi-omics (metabolomics and proteomics, with support from RNAseq) is performed over the time course of the CHO bioprocess producing an IgG1 mAb in 5 L bioreactors. Pathway analysis reveals that insufficient levels of Cys in the feed lead to Cys depletion in the cell. This depletion negatively impacts antioxidant molecules, such as glutathione (GSH) and taurine, leading to oxidative stress with multiple deleterious cellular effects. In this paper, the resultant ER stress and subsequent apoptosis that affects cell viability and viable cell density has been considered. Key metabolic enzymes and metabolites are identified that can be potentially monitored as the process progresses and/or increased in the cell either by nutrient feeding or genetic engineering. This work reinforces the centrality of redox balance to cellular health and success of the bioprocess as well as the power of multi-omics to provide an in-depth understanding of the CHO cell biology during biopharmaceutical production.


Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Cisteína/farmacologia , Animais , Anticorpos Monoclonais/efeitos dos fármacos , Reatores Biológicos , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Cisteína/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa/química , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Taurina/química
3.
Biotechnol J ; 13(10): e1700745, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29521466

RESUMO

As the demand for biological therapeutic proteins rises, there is an increasing need for robust and highly efficient bioprocesses, specifically, maximizing protein production by controlling the cellular nutritional and metabolic needs. A comprehensive lipidomics analysis has been performed, for the first time, over the time course of CHO cells producing an IgG1 monoclonal antibody (mAb) with fed batch 5 L bioreactors. The dynamic nature and importance of the CHO lipidome, especially on cellular growth and specific productivity, is demonstrated. A robust LC-MS method using positive and negative mode ESI was developed for lipid identification and quantitation of 377 unique lipids. The analysis revealed large changes in lipid features between the different days in bioprocessing including accumulation of triacylglycerol (TG) and lysophospholipid species with depletion of diacylglycerol (DG) species. Exploring pathway analysis where the lipid data was combined with polar metabolites and transcriptomics (RNA sequencing) revealed differences in lipid metabolism between the various stages of cellular growth and highlighted the role of key features of lipid metabolism on cell growth and specific productivity. The study demonstrates the importance of lipidomics in the expanding role of 'Omics methodologies in gaining insight into cellular behavior during protein production in a fed batch bioprocess.


Assuntos
Anticorpos Monoclonais/biossíntese , Reatores Biológicos , Técnicas de Cultura de Células , Meios de Cultura , Lipídeos/química , Animais , Técnicas de Cultura Celular por Lotes , Células CHO , Proliferação de Células , Cricetulus , Meios de Cultura/química , Imunoglobulina G/biossíntese , Lipídeos/análise
4.
Curr Opin Biotechnol ; 53: 151-157, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29414073

RESUMO

Chinese hamster ovary (CHO) cells have been extensively used for industrial production of biotherapeutics. With advances in cell line development and process optimization, production levels of therapeutic proteins using the CHO expression system have increased to beyond 10g per liter scale. These high-titer processes could challenge the secretory capacity of CHO cells, which can result in degradation and aggregation of the protein of interest. This review discusses bottlenecks in the secretory pathway of CHO cells that lead to inefficient secretion and aggregation of proteins, and summarizes current strategies to tackle these bottlenecks. In addition, emerging technologies that facilitate better understanding of cellular mechanisms in protein production could provide new avenues to improve the secretion and quality of protein therapeutics.


Assuntos
Agregados Proteicos , Proteínas Recombinantes/metabolismo , Via Secretória , Animais , Biotecnologia , Técnicas de Cultura de Células , Proteínas Recombinantes/uso terapêutico
5.
Anal Chem ; 89(24): 13097-13104, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29156137

RESUMO

Annotation of metabolites remains a major challenge in liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics. The current gold standard for metabolite identification is to match the detected feature with an authentic standard analyzed on the same equipment and using the same method as the experimental samples. However, there are substantial practical challenges in applying this approach to large data sets. One widely used annotation approach is to search spectral libraries in reference databases for matching metabolites; however, this approach is limited by the incomplete coverage of these libraries. An alternative computational approach is to match the detected features to candidate chemical structures based on their mass and predicted fragmentation pattern. Unfortunately, both of these approaches can match multiple identities with a single feature. Another issue is that annotations from different tools often disagree. This paper presents a novel LC-MS data annotation method, termed Biologically Consistent Annotation (BioCAn), that combines the results from database searches and in silico fragmentation analyses and places these results into a relevant biological context for the sample as captured by a metabolic model. We demonstrate the utility of this approach through an analysis of CHO cell samples. The performance of BioCAn is evaluated against several currently available annotation tools, and the accuracy of BioCAn annotations is verified using high-purity analytical standards.


Assuntos
Metabolômica/métodos , Metabolômica/normas , Animais , Células CHO , Células Cultivadas , Cromatografia Líquida , Cricetulus , Espectrometria de Massas em Tandem
6.
Biotechnol Prog ; 32(1): 74-82, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26560839

RESUMO

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.


Assuntos
Técnicas de Cultura de Células/métodos , Cinurenina/análogos & derivados , Oxirredução , Triptofano/metabolismo , Cromatografia Líquida , Meios de Cultura/química , Cinurenina/química , Cinurenina/metabolismo , Espectrometria de Massas , Metabolismo , Fotólise , Riboflavina/química , Triptofano/química
7.
Biotechnol Prog ; 31(5): 1201-11, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25919541

RESUMO

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi-prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench-top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10-fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening.


Assuntos
Técnicas de Cultura de Células , Regulação da Expressão Gênica , Proteínas Recombinantes/biossíntese , Animais , Automação , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Método de Monte Carlo
8.
Biotechnol Prog ; 29(6): 1519-27, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23926083

RESUMO

A 13-day fed-batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell-specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability.


Assuntos
Técnicas de Cultura Celular por Lotes , Técnicas de Cultura de Células/métodos , Imunoglobulina G/biossíntese , Mitocôndrias/metabolismo , Animais , Reatores Biológicos , Células CHO , Linhagem da Célula , Cricetulus , Glucose/metabolismo , Ácido Láctico/metabolismo , Consumo de Oxigênio
9.
Biotechnol Bioeng ; 109(10): 2523-32, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22473825

RESUMO

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench-scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody.


Assuntos
Anticorpos Monoclonais/metabolismo , Biotecnologia/métodos , Sulfetos/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Cisteína/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo
10.
Biotechnol Bioeng ; 103(3): 500-12, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19241387

RESUMO

Acetate is present in lignocellulosic hydrolysates at growth inhibiting concentrations. Industrial processes based on such feedstock require strains that are tolerant of this and other inhibitors present. We investigated the effect of acetate on Saccharomyces cerevisiae and show that elevated acetate concentrations result in a decreased specific growth rate, an accumulation of cells in the G1 phase of the cell cycle, and an increased cell size. With the cytostat cultivation technology under previously derived optimal operating conditions, several acetate resistant mutants were enriched and isolated in the shortest possible time. In each case, the isolation time was less than 5 days. The independently isolated mutant strains have increased specific growth rates under conditions of high acetate concentrations, high ethanol concentrations, and high temperature. In the presence of high acetate concentrations, the isolated mutants produce ethanol at higher rates and titers than the parental strain and a commercial ethanol producing strain that has been analyzed for comparison. Whole genome microarray analysis revealed gene amplifications in each mutant. In one case, the LPP1 gene, coding for lipid phosphate phosphatase, was amplified. Two mutants contained amplified ENA1, ENA2, and ENA5 genes, which code for P-type ATPase sodium pumps. LPP1 was overexpressed on a plasmid, and the growth data at elevated acetate concentrations suggest that LPP1 likely contributes to the phenotype of acetate tolerance. A diploid cross of the two mutants with the amplified ENA genes grew faster than either individual haploid parent strain when 20 g/L acetate was supplemented to the medium, which suggests that these genes contribute to acetate tolerance in a gene dosage dependent manner.


Assuntos
Acetatos/farmacologia , Adaptação Biológica , Resistência a Medicamentos , Inibidores do Crescimento/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animais , Etanol/metabolismo , Etanol/farmacologia , Dosagem de Genes , Perfilação da Expressão Gênica , Temperatura Alta , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidato Fosfatase/biossíntese , Fosfatidato Fosfatase/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/genética , Regulação para Cima
11.
Biotechnol Bioeng ; 102(1): 221-31, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18781685

RESUMO

Rational genetic alterations of a microorganism for a specific purpose are not possible in many situations where our knowledge of the relationship between phenotype and genotype is limited. In such cases evolutionary techniques must be applied. Evolutionary methods are usually time consuming; therefore, more efficient techniques are highly desirable. In this work we present the optimization of strain development in a cytostat. The time required for mutant strain isolation is dependent on the total cells present, the wild-type specific growth rate, the beneficial mutation probability, the mutant specific growth rate, and several bioreactor operating conditions. These parameters are highly related, and a theoretical model, as developed here, is needed to define the conditions that optimize the isolation. The model is based on a discrete, stochastic description of mutant formation and selection in the background of abundant wild-type cells. Using the model, we determined the optimal cytostat operating strategy for mutant isolation that varies according to the probability of beneficial mutations. It is also shown that mutants with as little as a 5% growth advantage can be isolated in less than 15 days which is significantly faster than in a chemostat. The described optimal mutant isolation procedure is expected to be particularly useful for the generation of industrial strains that are robust in challenging growth conditions.


Assuntos
Reatores Biológicos/microbiologia , Modelos Teóricos , Mutação , Evolução Molecular , Método de Monte Carlo
12.
Appl Environ Microbiol ; 74(22): 6880-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18836009

RESUMO

Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA](-) reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA](2-), the product of [Co(III)-EDTA](-) respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO(4). We demonstrate that [Co(III)-EDTA](-) reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation.


Assuntos
Cobalto/metabolismo , Shewanella/efeitos dos fármacos , Shewanella/metabolismo , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Cobalto/toxicidade , Citocromos c/metabolismo , Sulfato de Magnésio/metabolismo , Oxirredução , Shewanella/crescimento & desenvolvimento
13.
J Biotechnol ; 126(2): 163-72, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16716427

RESUMO

In a cytostat, a continuous culture is monitored and controlled by an automated flow cytometer system, based on the determination of the cell concentration and the single cell property distribution of the growing cell population. The growing culture can be maintained at steady state even at such low cell concentrations that the bioreactor medium composition is negligibly changed by the few cells. Therefore, the cell environment is precisely defined by the feed composition since products of cell growth are not present in significant amounts. Effects on cell growth of nutrients, of toxic compounds such as drugs, or of products made by the cells, if added to the feed medium, can be readily isolated. Using the cytostat, it is shown here that ethanol assumes the triggering function for the increase in cell size in Saccharomyces cerevisiae normally only seen at critical growth rates above critical cell densities. This suggests that ethanol assumes a quorum sensing function on cell growth when a critical cell density is reached.


Assuntos
Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/instrumentação , Microfluídica/instrumentação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Técnicas de Cultura de Células/métodos , Crescimento Celular , Fenômenos Fisiológicos Celulares , Proliferação de Células , Ambiente Controlado , Desenho de Equipamento , Análise de Falha de Equipamento , Etanol/metabolismo , Microfluídica/métodos
14.
Tokyo; United Nations University Press; c1996. xviii,282 p. ilus, mapas.
Monografia em Inglês | CidSaúde - Cidades saudáveis | ID: cid-54221
15.
Oxford; Oxford University Press; 1992. 331 p. tabs.
Monografia em Inglês | CidSaúde - Cidades saudáveis | ID: cid-15815
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