Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high ...quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future.
A novel type of Chinese hamster ovary cell mutant has been isolated with a constitutive defect in the synthesis of lipid-linked oligosaccharides. This mutant, designated AS15-1, incorporates 30-fold ...less glucosamine into an oligosaccharide-lipid fraction than the wild type. A gel filtration analysis has shown that a small amount of oligosaccharide-lipid corresponding to Man5GlcNAc2-lipid is formed in the mutant. This mutant shows temperature sensitivity for both growth and adhesion to substratum, and constitutively secretes several unusual proteins in large amounts.
Ornithine was found to be toxic to cells that lack ornithine aminotransferase (EC 2.6.1.13). These cells also accumulated substantial amounts of ornithine. The addition of methionine to these cells ...protected them from the toxic effects of ornithine and also diminished their accumulation of ornithine. Cells that have an active ornithine aminotransferase were resistant to the toxic effects of ornithine, and accumulated less of this compound.
External ATP causes a great increase in passive permeability to phosphorylated metabolites in several transformed cells, but not in untransformed cells. We have previously demonstrated that the ...external ATP-dependent permeability change was induced in Chinese hamster ovary cells, CHO-K1, only in the presence of a mitochondrial inhibitor (rotenone, KCN) or a cytoskeleton-attacking agent, vinblastine (Kitagawa, T. and Akamatsu, Y. Biochim. Biophys. Acta 649, 76-82 (1981); 734, 25-32 (1983. A similar ATP-dependent permeability change was also induced in CHO cells when the cells were treated with 10-30 microM trifluoperazine. This permeability change, like the previously mentioned ones, was found to be reversible and the treated cells remained viable. The permeability change induced by ATP and trifluoperazine was independent of changes in cellular ATP concentration and this property was the same as that of the permeability change with external ATP and vinblastine. Since trifluoperazine is known to interact with calmodulin and to inhibit calmodulin-dependent cellular functions, these results may indicate that calmodulin associated with the cytoskeleton plays an important role in control of the permeability change, although nonspecific perturbation by the drug of the membranes cannot be ruled out. Chlorpromazine and a naphthalene sulfonamide, W-7, also induced an ATP-dependent permeability change. However, these drugs, like mitochondrial inhibitors, reduced the cellular ATP concentration to induce the permeability change. Thus, a clear difference in the action of these drugs in intact cells was also shown in this study. Possible mechanisms for the ATP-dependent permeability change in mammalian cells are discussed.
Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased ...product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes.
Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated ...AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction. This was confirmed in mutant CHO Pro-5 cells deficient in the enzymes involved in glycoprotein biogenesis, as well as lectin interference studies. Binding of AAV9 to glycans with terminal galactose was demonstrated via glycan binding assays. Co-instillation of AAV9 vector with neuraminidase into mouse lung resulted in exposure of terminal galactose on the apical surface of conducting airway epithelial cells, as shown by lectin binding and increased transduction of these cells, demonstrating the possible utility of this vector in lung-directed gene transfer. Increasing the abundance of the receptor on target cells and improving vector efficacy may improve delivery of AAV vectors to their therapeutic targets.
Acyl-CoA:cholesterol acyltransferase from Chinese hamster ovary (CHO) cells was solubilized by deoxycholate, and then reconstituted in phosphatidylcholine/cholesterol liposomes. This reconstituted ...activity was totally dependent upon the cholesterol content of the mixture and showed saturation for cholesterol. Analysis of the reconstituted enzyme on linear Ficoll gradients shows that the enzyme has been incorporated into phospholipid/cholesterol liposomes. The CHO cell enzyme activity as measured by conventional assay (using cellular cholesterol as the substrate) was activated approximately 20-fold by low density lipoprotein. This activation process was independent of protein synthesis. When the above cell homogenates were assayed after optimal reconstitution, the activation produced by low density lipoprotein was essentially completely abolished. There was also no change in enzyme activity measured after reconstitution when cells were switched from sterol-containing medium to sterol-free medium, in contrast to a more than 7-fold drop in enzyme activity when assayed without reconstitution. These results suggest that the enzyme activity in intact cells is controlled by the content and composition of cellular lipids associated with the enzyme molecule. Since the intracellular messenger of low density lipoprotein is known to be cholesterol, it is likely that this enzyme activity in intact cells is primarily controlled by the cholesterol content in the vicinity of the enzyme molecule.
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