Moisant, Rémy (2024)
Toward a robust lipid supplementation for SP2/0 fed-batch culture: Improving process performance by leveraging lipoprotein variability.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00026549
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

In the context of recombinant monoclonal antibody production, the utilization of hybridoma cells is challenging due to their lipid auxotrophy. Although substituting serum with lipoproteins for lipid supplementation in SP2/0 cells has mitigated certain limitations associated with serum utilization, it has not addressed all the obstacles. The inherent variability in lipoprotein batches, derived from animal sources, exerts a significant impact on cell growth and the overall efficiency of the upstream cell culture process. This research aims to elucidate the relationship between lipoprotein batch-to-batch variability and the performance of upstream cell culture processes. An investigation was conducted utilizing multiple lipoprotein supplement batches from the same supplier. Notably, certain batches exhibited low performance characteristics during cell culture processes, manifesting as apoptosis induction and premature viability decline during fed-batch production. A physicochemical characterization of the lipoprotein supplement confirmed that lipoproteins predominantly contain sterols, fatty acids, and apolipoproteins, with a notable proportion of these constituents being oxidized. The induction of controlled oxidation in lipoprotein supplements resulted in observable changes, including browning of the supplement and increased UV absorbance, which, in turn, correlated with diminished performance in cell culture processes. This insight enabled the establishment of a threshold absorbance level at 276 nm, facilitating the identification of oxidized and low-performing lipoprotein supplement batches. This method serves to preemptively detect and exclude low-performing lipoprotein batches from the manufacturing process, thereby enhancing production robustness. Notably, it was determined that the chemical compounds responsible for viability drops were already present in the bovine serum raw material and were not introduced during the transformation process into lipoprotein supplements. To overcome limitations caused by lipoproteins, chemically defined lipid supplements were formulated. Utilizing methyl-beta-cyclodextrin inclusion complexes, fatty acids, and cholesterol were effectively solubilized in cell culture media. Optimization of the lipid-cyclodextrin balance, as well as fatty acid and cholesterol ratios, was undertaken to match the lipid requirements of SP2/0 cells while mitigating any cytotoxic effects stemming from cyclodextrin utilization. Although the cyclodextrin inclusion complexes with lipids demonstrated the potential to replace lipoproteins in the process, they yielded inferior performance, characterized by early viability decline and reduced overall growth. Hence, this alternative did not prove to be a viable substitute for lipoprotein supplementation, highlighting the challenges that persist in achieving chemically defined lipid supplementation in SP2/0 cell cultures with performance comparable to lipoprotein supplementation. Various other compounds were explored to support cell growth, with Trolox emerging as a promising candidate, effectively mitigating oxidation-related issues. These results should help to further optimize the formulation of cell culture media and identify parameters that influence the growth and production performance of cells for recombinant antibody production.

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