Browsing by Subject "Mammalian cell culture"

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Application of a PAT/QbD concept onto a Pharmaceutical Bioprocess
(2025) Graf, Alexander; Schaum, Alexander
In 2022 close to a hundred billion units of pharmaceuticals were sold in Germany alone (Radtke, 2023). Patient safety and high efficacy are the most critical factors in producing these drugs. This results in a need for manufacturers to constantly output high-quality products. Consequently, there has been an ongoing adoption of Quality by Design (QbD) and Process Analytical Technologies (PAT) in the industry over the last decades. This thesis focused on advancing different spectroscopic methods as PAT tools in the context of QbD. First, a novel 2D-fluorescence (2DF) sensor was investigated for its usability in-process monitoring of mammalian cell cultures – qualitative and quantitative. Second, Raman spectroscopy was examined for its use in bioprocess development and as a control device in production bioreactors. In the first part of this work, the 2DF technology demonstrated its versatility as, on the one hand, an effective method for golden batch monitoring, i.e., for promptly detecting deviations within the process. On the other hand, the fluorescence signals can be correlated to cell count and viability, making it a suitable in-line alternative to traditional off-line cell counting. In the context of QbD, fluorescence spectroscopy can furthermore give the user more insight into the cellular metabolism, as, for example, co-enzymes like NADH can be detected. The second part focuses on Raman spectroscopy as a valuable tool during process development and for in-line process control of critical process parameters. First, a Raman spectrometer was integrated into two automated mini-bioreactor systems – one with 15 mL and the other with 250 mL single-use vessels. These systems are commonly used in cell line development and upstream process development campaigns, especially for economic execution of Design of Experiment studies. Integrating a Raman spectrometer in these highly automated systems made it possible to efficiently generate a large Raman dataset for robust modeling of several essential process parameters, such as glucose, lactate, glutamine, glutamate, and target protein titer. In the case of the glucose model, scale-up to a 50 L bioreactor was successfully made for in-line monitoring of said parameter. Finally, Raman spectroscopy was integrated into a perfusion process. In combination with a biocapacitance probe for in-line cell count control, the Raman system was successfully utilized for on-line control of the glucose concentration. This paper proves that PAT sensors can be utilized as enablers for process intensification and, consequently, as a step toward continuous processing.