Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flowfiltration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultrahigh seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 10 6 cells/ mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime and with comparable product quality compared to low-seeded cultures. Driven by mechanistic modelling and next-generation sequencing (NGS) the process had been optimized by selecting the media composition in a way that minimized cellular adaptation between perfusion and production culture. As a main feature, lactate feeding was applied in the intensified approach to promote cell culture performance and process scalability was proven via transfer to pilot-scale i.e., 20 L pre-stage perfusion and 80 L production reactor. Moreover, an earlier shift from a growth associated to a production stage associated gene expression pattern was identified for uHSD cultures compared to the reference. Overall, we showed that the described intensification strategy yielded in a higher volumetric productivity and is applicable for existing or already approved molecules in common, commercial fed-batch facilities. This work provides an in-depth molecular understanding of cellular processes that are detrimental during process intensification.

A radioimmunoassay for the immunosuppressant drug Cyclosporin A has been developed which makes possible the monitoring of the drug by direct measurements in clinical plasma and serum samples. The antisera have been produced in rabbits using the hemisuccinate derivative of a structural analogue of Cyclosporin A as a hapten. The assay has both adequate specificity and sensitivity for Cyclosporin A to be suitable for the routine monitoring of therapy. Some degree of cross-reactivity has been shown to occur with four metabolites which were isolated from urine samples.

The goal of this study is to develop a macroscopic mechanistic model describing growth and production within fed-batch cultivations of CHO cells. The model should be used for process characterization as well as for process monitoring including real-time parameter adaptations. The model proved to be able to describe a data-set of 40 processes differing in clones, scales, and process conditions with a normalized root mean square error of approximately 10%. However, due to limited parameter identifiability and limited knowledge about physiologically meaningful parameter values, a broad range of parameters could describe the data with similar quality. This hampered comparison of the model parameters as well as their real-time estimation. Therefore an iterative workflow combining techniques like sensitivity and identifiability analysis, analysis of the specific rates as well as structural adaptations of the parameter space is developed. By applying it the parameter variability could be reduced by 80% with similar predictive power as the original parameters. Summing up, based on a mechanistic CHO model, a generic and transferrable workflow is created for target-oriented parameter estimation in case of limited parameter identifiability. Finally, we suggest a methodology, which fits ideally into the frame of Process Analytical Technology aiming to increase process understanding.

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