High density CHO cell cultures: Improved productivity …

Several early observations (they are hardly "conclusions") emergefrom research conducted thus far. The notion that certain parts of Sicily stillgenetically reflect the influence of specific ancient peoples (Phoenicians, Greeks) has beenlargely disproven, yet certain small, relatively-isolated towns seem to be markedby a predominance of one medieval group or another (Arab, Norman). Leaving asidespecialized studies, if we consider the major Y haplogroups, Sicily'spopulation-genetic distribution is somewhat similar (though by no meansidentical) to mainland Italy's. If only approximately the proportions are: JGroup (J1, J2, etc.) 35%, R Group (primarily R1b) 25%, I Group 15%, K Group 10%,H Group 10%, Others (E, T, G, etc.) 5%. Along female lines, Sicilians' descent from the seems to be distributed fairly equally, but much more data mustbe collected in this area. These factors (and scholarly studies) all point to theisland's multi-peopling as the main cause of its genetic diversity.

What is more, the effect of cell-cell crosstalk depended on the type of culture system.

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The characterised proteins were sterilised andused to coat 96 well plates for cell culture.

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The first experiments in tissue culture in animals were carried out in 1907 by R. Garrison: germs cells of the frog embryo nervous system have been remaining alive for a few weeks in a small amount of lymph, with continuous grows of new fibers. Further progress in the development of tissue culture were mainly due to the creation and improvement of synthetic nutrient medium containing the materials necessary to keep the cells alive.

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Continual growth in the biopharmaceutical industry in recombinant protein production has resulted in an ever increasing demand for optimised production processes, particularly in the area of upstream mammalian cell culture bioprocessing. The inefficient energy demands of mammalian cells in culture persists as a limiting factor for optimisation of cell culture processes. The Process Analytical Technology (PAT) framework initiative, put forward by the FDA in 2001, outlines measures for the strict monitoring and control of critical process parameters (CPP-s) affecting cell growth, productivity and product quality. The identification of CPP-s affecting protein quality (namely glycosylation and/or aggregation) has never been more important in seeking recombinant protein drug approval. A project was designed aimed at addressing the issue of CPP identification in high density CHO cell cultures producing the recombinant protein IgG1. Initial studies identified the importance of L-glutamine for cell growth and productivity, but also for increased complexity of the glycoform on the IgG1 protein. This was identified by comparing cultures containing 4 mM L-Glutamine to cultures containing 0 and 4 mM L-glutamate. Interestingly this study also identified that the impact of the by-product ammonia on recombinant protein quality may be over-estimated by investigating such effects through the incorporation of high initial ammonia concentrations (~15 mM). In addressing the title of the project, encapsulation as a mode of cultivation for suspension adapted mammalian cells was also investigated, with significantly increased yields in cell (3.7-fold) and product titres being achieved for batch encapsulated cultures in comparison to suspension cultures. Research to date has failed to identify if encapsulation may have implications on protein quality. Initial studies identified that the quality of the protein harvested at the end of the stationary growth period in both batch and suspension cultures was relatively similar. In order to further increase the maximum cell yields and product titres obtained in an encapsulated culture, a control-fed perfusion strategy was designed and applied to the encapsulated cells. Further increased cell yields, 10-fold higher than that which was achieved in a suspension batch cultures were noted. The volumetric titre of recombinant protein in the control-fed perfusion cultures was determined to be ~2.65- fold higher than that achieved in the batch encapsulated cultures. The rIgG1 had a higher degree of complexity at the end of the extended growth period in the control-fed perfusion culture in comparison to batch encapsulated cultures. The importance of monitoring cell viability and optimising the rate of perfusion was noted by the occurrence of a decrease in glycan complexity, associated with the accumulation of dead cells and glycosidase release.

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However, the direct comparison of these cells demonstrated limited similarity of cell lines to the primary human osteoblasts indicating that their use should be limited to appropriate and specific research questions.