Quality-by-Design (QbD) is demanded by regulatory authorities in biopharmaceutical production. Within the QbD frame advanced process control (APC), facilitated through process analytical technology (PAT) and digital twins (DT), plays an increasingly important role as it can help to assure to stay within the predefined proven acceptable range (PAR).This ensures high product quality, minimizes failure and is an important step towards a real-time-release testing (RTRT) that could help to accelerate time-to-market of drug substances, which is becoming even more important in light of dynamical pandemic situations. The approach is exemplified on scFv manufacturing in Escherichia coli. Simulation results from digital twins are compared to experimental data and found to be accurate and precise. Harvest is achieved by tangential flow filtration followed by product release through high pressure homogenization and subsequent clarification by tangential flow filtration. Digital twins of the membrane processes show that shear rate and transmembrane pressure are significant process parameters, which is in line with experimental data. Optimized settings were applied to 0.3 bar and a shear rate of 11,000 s−1. Productivity of chromatography steps were 5.3 g/L/d (Protein L) and 2167 g/L/d (CEX) and the final product concentration was 8 g/L. Based on digital twin results, an optimized process schedule was developed that decreased purification time to one working day, which is a factor-two reduction compared to the conventional process schedule. This work presents the basis for future studies on advanced process control and automation for biologics production in microbials in regulated industries.