In bioprocess engineering the Qualtiy by Design (QbD) initiative encourages the use of models to define design spaces. However, clear guidelines on how models for QbD are validated are still missing. In this review we provide a comprehensive overview of the validation methods, mathematical approaches, and metrics currently applied in bioprocess modeling. The methods cover analytics for data used for modeling, model training and selection, measures for predictiveness, and model uncertainties. We point out the general issues in model validation and calibration for different types of models and put this into the context of existing health authority recommendations. This review provides a starting point for developing a guide for model validation approaches. There is no one-fits-all approach, but this review should help to identify the best fitting validation method, or combination of methods, for the specific task and the type of bioprocess model that is being developed.

Optical density (OD) is a critical process parameter during fermentation, this being directly related to cell density, which provides valuable information regarding the state of the process. However, to measure OD, sampling of the fermentation broth is required. This is particularly challenging for high-throughput-microbioreactor (HT-MBR) systems, which require robotic liquid-handling (LiHa) systems for process control tasks, such as pH regulation or carbon feed additions. Bioreactor volume is limited and automated at-line sampling occupies the resources of LiHa systems; this affects their ability to carry out the aforementioned pipetting operations. Minimizing the number of physical OD measurements is therefore of significant interest. However, fewer measurements also result in less process information. This resource conflict has previously represented a challenge. We present an artificial neural-network-based soft sensor developed for the real-time estimation of the OD in an MBR system. This sensor was able to estimate the OD to a high degree of accuracy (>95%), even without informative process variables stemming from, e.g., off-gas analysis only available at larger scales. Furthermore, we investigated and demonstrated scaling of the soft sensor’s generalization capabilities with the data from different antibody fragments expressing Escherichia coli strains. This study contributes to accelerated biopharmaceutical process development.

During recombinant protein production with E. coli, the integrity of the inner and outer membrane changes, which leads to product leakage (loss of outer membrane integrity) or lysis (loss of inner membrane integrity). Motivated by current Quality by Design guidelines, there is a need for monitoring tools to determine leakiness and lysis in real-time. In this work, we assessed a novel approach to monitoring E. coli cell integrity by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Various preprocessing strategies were tested in combination with regression (partial least squares, random forest) or classification models (partial least squares discriminant analysis, linear discriminant analysis, random forest, artificial neural network). Models were validated using standard procedures, and well-performing methods were additionally scrutinized by removing putatively important features and assessing the decrease in performance. Whereas the prediction of target compound concentration via regression was unsuccessful, possibly due to a lack of samples and low sensitivity, random forest classifiers achieved prediction accuracies of over 90% within the datasets tested in this study. However, strong correlations with untargeted spectral regions were revealed by feature selection, thereby demonstrating the need to rigorously validate chemometric models for bioprocesses, including the evaluation of feature importance.

Intermittent bolus feeding for E. coli cultivations in minibioreactor systems (MBRs) profoundly affects the cell metabolism. Bolus feeding leads to temporal substrate surplus and transient oxygen limitation, which triggers the formation of inhibitory byproducts. Due to the high oxygen demand right after the injection of the substrate, the dissolved oxygen tension (DOT) signal exhibits a negative pulse. This contribution describes and analyzes this DOT response in E. coli minibioreactor cultivations. In addition to gaining information on culture conditions, a unique response behavior in the DOT signal was observed in the analysis. This response appeared only at a dilution ratio per biomass unit higher than a certain threshold. The analysis highlights a plausible relationship between a metabolic adaptation behavior and the newly observed DOT signal segment not reported in the literature. A hypothesis that links particular DOT segments to specific metabolic states is proposed. The quantitative analysis and mechanistic model simulations support this hypothesis and show the possibility of obtaining cell physiological and growth parameters from the DOT signal.