McNamara_2016_characterisation.pdf (2.78 MB)
Characterisation of the fundamental function(s) of bovine serum albumin in the culture of NS0 cells
thesisposted on 2022-08-25, 14:36 authored by Liam McNamara
The large-scale production of therapeutic monoclonal antibodies (mAbs) is commonly carried out using NS0 cells, which are a cholesterol deficient mouse myeloma cell line. The non-ionic surfactant Pluronic F-68 is routinely added to large-scale cell cultures to protect cells from damage experienced in the bioreactor, however there have been reports detailing limitations of Pluronic F-68. Bovine Serum Albumin (BSA) is also added to media used for the large-scale cell culture of some cell lines (including NS0 cells); however there has been limited research published to support the function of BSA in large-scale cell culture. BSA has been suggested to have a number of possible functions in large-scale cell culture media, including; i) offering protection against damage experienced by cells in the bioreactor, ii) acting as a carrier of nutrients (e.g. cholesterol) and iii) acting as an antioxidant. The addition of BSA to media increases the risk of contamination with adventitious agents and requires additional controls by regulatory agencies to ensure patient safety. In this project two hydromechanical stress models (moderate and extreme hydromechanical stress) were developed in baffled shake flasks to investigate the effects of BSA on NS0 cells. High concentrations of an effective lot of Pluronic F-68 (1g/L) completely protected the NS0 cells in both hydromechanical stress models irrespective of the presence of BSA. The results showed that BSA protects NS0 cells from damage in both baffled shake flask models in media with low concentrations of Pluronic F-68 (≤0.1g/L). Although BSA was shown to offer protection to NS0 cells experiencing laminar shear stress, BSA offered greater protection to the cells in the hydromechanical stress models. This suggests that BSA may have a number of different protective mechanisms of action for NS0 cells experiencing damage. The protective effect of BSA was shown to be rapidly afforded and lost in both baffled shake flask models, suggesting that BSA offers physical or fast-acting biological protection for NS0 cells experiencing hydromechanical stress. Investigations into the possible physical or fast-acting biological protective mechanism(s) of action of BSA (in media with reduced concentrations of Pluronic F-68) showed that the BSA reduces the surface tension of the NS0 cell culture medium. The protection afforded by BSA was also shown to be independent of cholesterol supplementation and protein tertiary structure (using denatured BSA). BSA was shown to coat the NS0 cells in culture and the NS0 cell membrane hydrophobicity was reduced in the presence of BSA. Methyl cellulose, a non-animal sourced surfactant, was shown to protect NS0 cells from hydromechanical stress similar to BSA, therefore the protective effect of BSA may be substituted by a non-animal sourced surfactant. The findings in this project show that BSA protects NS0 cells from damage by a number of physical or fast-acting biological mechanisms when the protective effect of Pluronic F-68 is limited. The results suggest that the large-scale cell culture of NS0 cells would be possible in media not supplemented with BSA if the protective effect of Pluronic F-68 is not limited (or if the protection afforded by BSA is replaced with a non-animal derived surfactant).
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