Comparison of mechanistic and hybrid modeling approaches for characterization of a CHO cultivation process: Requirements, pitfalls and solution paths.

Despite the advantages of mathematical bioprocess modeling, successful model implementation already starts with experimental planning and accordingly can fail at this early stage. For this study, two different modeling approaches (mechanistic and hybrid) based on a four-dimensional antibody-producing CHO fed-batch process are compared. Overall, 33 experiments are performed in the fractional factorial four-dimensional design space and separated into four different complex data partitions subsequently used for model comparison and evaluation.

Fundamental insights in early-stage inclusion body formation.

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.

Model Transferability and Reduced Experimental Burden in Cell Culture Process Development Facilitated by Hybrid Modeling and Intensified Design of Experiments.

Reliable process development is accompanied by intense experimental effort. The utilization of an intensified design of experiments (iDoE) (intra-experimental critical process parameter (CPP) shifts combined) with hybrid modeling potentially reduces process development burden. The iDoE can provide more process response information in less overall process time, whereas hybrid modeling serves as a commodity to describe this behavior the best way.

Proton-transfer-reaction mass spectrometry (PTR-MS) for online monitoring of glucose depletion and cell concentrations in HEK 293 gene therapy processes.

Objectives: The applicability of proton-transfer-reaction mass spectrometry (PTR-MS) as a versatile online monitoring tool to increase consistency and robustness for recombinant adeno-associated virus (rAAV) producing HEK 293 bioprocesses was evaluated. We present a structured workflow to extract process relevant information from PTR-MS data.

Results: Reproducibility of volatile organic compound (VOC) measurements was demonstrated with spiking experiments and the process data sets used for applicability evaluation consisted of HEK 293 cell culture triplicates with and without transfection.

Soft sensor based on 2D-fluorescence and process data enabling real-time estimation of biomass in cultivations.

In bioprocesses, specific process responses such as the biomass cannot typically be measured directly on-line, since analytical sampling is associated with unavoidable time delays. Accessing those responses in real-time is essential for Quality by Design and process analytical technology concepts. Soft sensors overcome these limitations by indirectly measuring the variables of interest using a previously derived model and actual process data in real time.

Hybrid Modeling and Intensified DoE: An Approach to Accelerate Upstream Process Characterization.

Process characterization is necessary in the biopharmaceutical industry, leading to concepts such as design of experiments (DoE) in combination with process modeling. However, these methods still have shortcomings, including large numbers of required experiments. The concept of intensified design of experiments (iDoE) is proposed, that is, intra-experimental shifts of critical process parameters (CPP) that combine with hybrid modeling to more rapidly screen a particular design space.

Comparison of Modeling Methods for DoE-Based Holistic Upstream Process Characterization.

Upstream bioprocess characterization and optimization are time and resource-intensive tasks. Regularly in the biopharmaceutical industry, statistical design of experiments (DoE) in combination with response surface models (RSMs) are used, neglecting the process trajectories and dynamics. Generating process understanding with time-resolved, dynamic process models allows to understand the impact of temporal deviations, production dynamics, and provides a better understanding of the process variations that stem from the biological subsystem.

Injection of the diaphragmatic crus with methylene blue for coloration of the canine thoracic duct.

Objective: To describe and compare onset and intensity of thoracic duct (TD) coloration after injection of methylene blue into the diaphragmatic crus and mesenteric lymph node.

Study Design: Experimental study.

Animals: Adult dogs (n = 18).