No abstract

Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals. However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells. This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia. Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis. It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK‐21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem. Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control. Moreover, a 1.4‐fold‐higher adenosine triphosphate (ATP) content was achieved. The flux of labeled [14C]‐glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation. Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 238–246, 1999.

No abstract

To delineate parameters contributing to the extracellular lifetime of retroviral vectors, we carried out stability tests of retroviruses derived from cell lines of different origin and kept under different cultivation conditions. Results show that amphotropic mouse retroviruses (MLV-A) derived from human and hamster cells exhibit 2- to 3-fold higher half-lives compared to retroviruses from mouse cells. Cultivation at 32 degrees C has been reported to yield high virus titers. However, the benefit of virus production in mouse cells at 32 degrees C is controversial. In our hands the cultivation temperature affected, hitherto not noticed, the half-life time of MLV-A. The 37/32 degrees C shift resulted in a 3-fold decrease of viral half-lifes compared to MLV-A released from mouse cells at 37 degrees C. Thus, MLV-A released at 37 degrees C is phenotypically different from MLV-A synthesized at 32 degrees C. Increased virus stability was inversely correlated with the level of cholesterol in the viral membrane. Finally, depletion of viral cholesterol in vitro resulted in intact virus with increased thermal stability. Thus, retrovirus lability depends on the host cell and parallels the cholesterol amount in the viral lipid shell.

Two size classes of 0-glycosidically linked oligosaccharides were liberated from glycoprotein El of mouse hepatitis virus (MHV) A59 by reductive fl-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Aca2-3 Galfll-)3 GaINAc) comprised 3507o of the total carbohydrate side chains, while the remaining 650%o of the oligosaccharides of El had the branched structure B:Neu5Aca2-*3 Gal,01-*3 (Neu5Aca2--6) GalNAc. Both oligosaccharides were linked to the El polypeptide via N-acetylgalactosamine, and 20%o of the sialic acids present in El glycopeptides were found to consist of N-acetyl-9-mono-0-acetylneuraminic acid. The reported structures of the 0-linked glycans are discussed in the context of the amino acid sequence of El, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential 0-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical 0-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

No abstract

No abstract

A quantitative and mechanistic understanding of intracellular transport processes in eukaryotic cells during transient transfection is an important prerequisite for the systematic and specific optimization of transient gene expression procedures for pharmaceutic and industrial protein production. There is evidence that intracellular transport processes during gene delivery and their regulation may have significant influence on the transfection efficiency. This contribution describes a compartmented, spatiotemporally resolved and stochastic modeling approach that describes intracellular transport processes responsible for gene delivery during transient transfection. It enables a detailed prediction and analysis and identification of potential bottlenecks. This model is currently being adapted to a model cell line, HEK293s. The simulated results are compared with experimental quantitative polymerase chain reaction (qPCR) data and confocal imaging data obtained with transfected and stained HEK293 cells. Global parameter estimation is performed to qPCR data based on two different novel plasmid constructs in order to identify candidates for plasmid-specific transport parameter variations. The influence of the specific property of HEK293 cells to grow in clusters is investigated and the impact of active microtubule transport depending on cell morphology and clustering is examined. A general sensitivity analysis allows for the identification of the sensitive parameters.