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Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A(280)) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A(280) was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recombinant therapeutic proteins produced in mammalian cells represent a major class of biopharmaceuticals. In recent years, their demand has increased dramatically and is now driving the development of a variety of improvements to maximize their expression in mammalian cells. Advances in media- and process optimization have already resulted in more than 100-fold improvement in yield, but further insights and subsequent targeted modifications with respect to the general biology of cells (genomics, physiology, selection and adaptation) in bioreactors are hoped to further improve protein yields and quality in the near future.:, (© 2008 Elsevier Ltd . All rights reserved.)

A new scalable reactor was developed by applying a novel mixing principle that allows the large-scale cultivation of mammalian cells simply with surface aeration using air owing to increased liquid-gas transfer compared to standard stirred-tank bioreactors. In the cylindrical vessels (50 mL-1500 L) with a helical track attached to the inside wall, the liquid moved upward onto the track as the result of orbital shaking to increase the liquid-gas interface area significantly. This typically resulted in a 5-10-fold improvement in the volumetric mass transfer coefficient (k(L)a). In a 1500-L helical track vessel with a working volume of 1000 L, a k(L)a of 10h(-1) was obtained at a shaking speed of 39 rpm. Cultivations of CHO cells in a shaken 55-L helical track bioreactor resulted in improved cell growth profiles compared to control cultures in standard systems. These results demonstrated the possibility of using these new bioreactors at scales of 1000 L or more.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.