The main goal in biopharmaceutical production is achieving high volumetric productivity while maintaining product quality (i.e. glycosylation). The objectives of this project were to explore the use of dielectric analysis in the early detection of cell demise and to analyze the impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and glycosylation.
Measurements of changes in the polarizability of individual cells can be performed in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked according to their polarizability and recorded as a force index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer. Discrete cell sub-populations were identified as cells passed through the various stages of apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in the mitochondrial metabolism linked with early apoptosis for both inducers.
In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination with 1 mM uridine or 1 mM uridine + 8 μM MnCl2 was added to the basal and feed medium for two CHO cell lines to determine their impact on the biopharmaceutical production and the glycosylation process. The results showed that the addition of all three precursors combined increased UDP-Gal, which increased and maintained the galactosylation index during the bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a further increase in the UDP-Gal concentration did not increase the galactosylation. A negative impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which was linked to the CHO-EG2 cell line being DHFR-/-.
This work presents a dielectric detection method to monitor early changes in the cell metabolism and information for shifting and maintaining galactosylation during biopharmaceutical production. / February 2016
| Identifer | oai:union.ndltd.org:MANITOBA/oai:mspace.lib.umanitoba.ca:1993/31080 |
| Date | January 2015 |
| Creators | Braasch, Katrin |
| Contributors | Butler, Michael (Microbiology), Court, Deborah (Microbiology) Levin, David (Biosystems Engineering) Kallos, Michael (University of Calgary) |
| Publisher | John Wiley and Sons, Springer |
| Source Sets | University of Manitoba Canada |
| Detected Language | English |
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