Continuous Process Verification 4.0 application in upstream: adaptiveness implementation managed by AI in the hypoxic bioprocess of the Pichia pastoris cell factory

The experimental approach developed in this research demonstrated how the cloud, the Internet of Things (IoT), edge computing, and Artificial Intelligence (AI), considered key technologies in Industry 4.0, provide the expected horizon for adaptive vision in Continued Process Verification (CPV), the final stage of Process Validation (PV). <i>Pichia pastoris</i> producing <i>Candida rugosa</i> lipase 1 under the regulation of the constitutive <i>GAP</i> promoter was selected as an experimental bioprocess. The bioprocess worked under hypoxic conditions in carbon-limited fed-batch cultures through a physiological control based on the respiratory quotient (<i>RQ</i>). In this novel bioprocess, a digital twin (DT) was built and successfully tested. The implementation of online sensors worked as a bridge between the microorganism and AI models, to provide predictions from the edge and the cloud. AI models emulated the metabolism of <i>Pichia</i> based on critical process parameters and actionable factors to achieve the expected quality attributes. This innovative AI-aided Adaptive-Proportional Control strategy (AI-APC) improved the reproducibility comparing to a Manual-Heuristic Control strategy (MHC), showing better performance than the Boolean-Logic-Controller (BLC) tested. The accuracy, indicated by the Mean Relative Error (MRE), was for the AI-APC lower than 4%, better than the obtained for MHC (10%) and BLC (5%). Moreover, in terms of precision, the same trend was observed when comparing the Root Mean Square Deviation (RMSD) values, becoming lower as the complexity of the controller increases. The successful automatic real time control of the bioprocess orchestrated by AI models proved the 4.0 capabilities brought by the adaptive concept and its validity in biopharmaceutical upstream operations.