Abstract Background: Recent regulatory initiatives encourage the use of Process Analytical Technologies (PAT) and Quality-by-Design (QbD) in order to gain a science- and risk-based understanding of bioprocesses. PAT and QbD are increasingly recognized as versatile means to characterize process parameters with respect to product quality. Aims: In this thesis, the application of QbD and PAT is demonstrated on the upstream processing of the production of recombinant human bone morphogenetic protein-2 (rhBMP-2), a pharmacologically relevant inductor of osteogenesis. The primary objective of this work is to elucidate physiological process parameters using dynamic experimentation and design-of-experiments (DoE). Material and Methods: For the fermentative production of rhBMP-2 a recombinant E. coli mixed-feed expression system under the control of the pBAD promoter is used. For the mixed feed strategy, L-arabinose is employed as inducer of the recombinant construct and D-glucose is used as main C source. Pulse and ramp experimets are conducted to elucidate physiological boundaries of the rhBMP-2 production. Soft-sensors as well as in-line measurements derived from FTIR are implemented as PAT tools to monitor and control process conduct. Design-of-experiments (3-factor CCF design) is performed to analyze the contribution and interaction of physiological process parameters. Results: Maximum metabolic conversion rates of L-arabinose were determined as a function of temperature and uptake of D-glucose using a novel dynamic method. With respect to critical quality attributes, IB purity and product titer, IB purity was found to be positively associated with qs gluc and the cultivation temperature whereas, product titer was only found to be positively associated with the temperature. Conclusion: In summary, the use of soft-sensor assisted dynamic experimentation and physiological design of experiments is successfully shown to characterize the contribution of physiological process parameters on the upstream processing of rhBMP-2 production.