Investigation of phenotypic population heterogeneity in industrial cell culture processes

In the production of biopharmaceuticals such as recombinant therapeutic antibodies using mammalian cells, phenotypic population heterogeneities can occur, potentially leading to unexpected bioprocess outcome in terms of robustness, growth, productivity or efficacy of the final product. To investigate the temporal dynamics of phenotypic population heterogeneity in mammalian cells (e.g. Chinese hamster ovary cells) on the single cell, fluorescent reporter constructs are transiently or stably integrated into the cell´s genome. Depending on the reporter structure, one or more fluorescent proteins are expressed by the cell, allowing non-invasive indication of the status of the cell, activity of cell organelles, e.g., mitochondria and autophagosome, or metabolism when the resulting fluorescence in monitored with flow cytometry analysis.

Unlike recent applications of reporter constructs, this project not only focuses on the cell´s reaction to well-defined and controlled cultivation conditions and bioprocess parameters, but also on phenotypic population heterogeneity occurring in industry-relevant and larger-scale bioreactor systems are assessed applying different scale-down bioreactor configurations.

Contact person: Dr.-Ing. Lukas Arndt

Multifactorial Analysis of HEK293-F Cell Aggregation: Uncovering the Role and Interactions of Bioprocess and Media Factors

Cell aggregation, the tendency of cells to form clusters, remains a challenge in mammalian cell culture. Especially the presence of large cell aggregates can negatively affect cell viability, transfection efficiency, and overall bioprocess performance. Although suspension-adapted cell lines and optimized culture media have reduced the occurrence of aggregation, aggregate formation is still observed in widely used production hosts such as HEK293-F. The underlying causes of this phenomenon are complex, involving interactions between bioprocess conditions, culture medium composition, and cellular responses.

This project focuses on improving the understanding of cell aggregation by systematically investigating the contribution of bioprocess and media related parameters and their interactions. While the effects of several factors have been reported individually, their combined influence on aggregation behavior remains poorly understood.

To address these knowledge gaps, a Design of Experiments (DoE) framework is employed in controlled stirred-tank bioreactor cultivations. Particular emphasis is placed on the interaction of established aggregation effectors as well as the identification of less-characterized factors. Cell aggregation behavior is quantified through temporal aggregate size distribution analysis and linked to bioprocess performance indicators including cell growth and viability.

Beyond experimental characterization, the project aims to establish a quantitative framework for describing aggregation phenomena across cultivation systems. The experimental data will provide the foundation for the development and parameterization of a mechanistic cell aggregation model, with the long-term goal of improving bioprocess understanding and development of dedicated control and mitigation approaches in mammalian cell culture bioprocessing.

Contact person: Niklas Nathusius