Escherichia coli is one of the most exploited organisms for industrial production of recombinant proteins using bioprocesses [1, 2]. Within process development, critical process parameters are identified and consequently investigated from lab to production scale. In order to minimize process development effort the generation of product independent, transferable prior process knowledge is of utmost interest. Currently, bioprocesses are commonly developed based on technical process parameters as e.g. volumetric feeding rates, generating hardly transferable, technology oriented process knowledge. After decades of focusing on technical parameters, a more physiological approach has emerged [3-6]. Structured in two parts, this thesis aims to establish and assess physiological bioprocess development as well as to analyze whether it bears significant advantages compared to conventional approaches. 1) Establishment and investigation of analytical methods to quantify and detect physiological processes and phenomena with respect to accuracy and robustness: - Cell lysis, as physiological event, features cytosolic protein release . For protein quantification in complex sample matrixes the error of the method was reduced from >200% to <50%. - High titer expression of protein frequently features the physiological phenomena of inclusion body (IB) formation. To analyze the IB growth as an effect of expression rates, the novel method of nano particle tracking analysis for IB sizing was established and successfully verified. - Physiological bioprocess control requires accurate biomass estimation. In the context of early bioprocess development, a weighted average combination of first principle soft sensors was proven the most suitable approach for real time biomass estimation. 2) Analysis of the advantages and challenges of physiological bioprocess development at hand of industrial relevant production processes. - Physiological bioprocess development requires single numerical descriptors of physiology representing distinct process phases. Therefore, a novel variable for physiological phase definition and a workflow to increase process knowledge integration was illustrated. - A physiological feeding strategy based on the specific substrate uptake rate (qS), in comparison to technological feeding profiles, was shown to be highly beneficial in terms of product titer. - Physiological process control requires the accurate definition of physiological limits e.g. the critical qS (qScrit). Using controlled oscillations of qS, qScrit was shown to be highly dependent on time after induction and on the average metabolic activity qSmean. The latter finding calls for technological strategies to cope with the dynamic nature of qScrit. - Using a combination of first principle softsensors a closed loop real time control approach of qS was established. Hereby, substrate accumulation in late process phases was effectively avoided. This thesis comprehensively discusses advantages and challenges of physiological bioprocess development. Physiologic process development grants deeper insights into relevant physiological processes and fosters the general understanding of the behavior of the production strain regardless of an associated titer increase. Physiological bioprocess control approaches, as the backbone of physiological bioprocess development are even able to substitute strain characterization experiments. It grants additional degrees of freedom and thereby potentially allows for higher time space yields. It can be concluded, that physiological bioprocess development asks for a one time effort investment for the establishment of sensitive analytics and physiological control approaches but on the long term it rewards with a substantial increase in information to effort ratio.