Abstract
ObjectivesThe 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.ResultsReproducibility 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. The developed data workflow enabled the identification of six VOCs, of which two were used to develop a soft sensor providing better real-time estimates than the conventional capacitance sensor. Acetaldehyde, another VOC, provides online process information about glucose depletion that can directly be used for process control purposes.ConclusionsThe potential of PTR-MS for HEK 293 cell culture monitoring has been shown. VOC data derived information can be used to develop soft sensors and to directly set up new process control strategies.
Highlights
Recombinant adeno-associated viruses are one of the most popular viral vectors for in vitro and in vivo gene delivery used to treat diseases derived from monogenic disorders (Naso et al 2017)
We describe a workflow to exclude irrelevant proton-transfer-reaction mass spectrometry (PTR-MS) masses and simultaneously identify valuable ones for Recombinant adeno-associated viruses (rAAV) producing HEK 293 cell culture processes by applying an unsupervised learning strategy combined with process knowledge
The experimental setup for online monitoring of volatile organic compound (VOC) consisted of a bioreactor, a sampling system, and a high-sensitivity hs-PTR-MS instrument (Ionicon Analytik, Austria)
Summary
Recombinant adeno-associated viruses (rAAV) are one of the most popular viral vectors for in vitro and in vivo gene delivery used to treat diseases derived from monogenic disorders (Naso et al 2017). Many efforts and resources have been focused on scalable manufacturing processes to achieve high titers with high full to empty capsid ratios. This emerging manufacturing technology is mostly based on triple transfection in HEK 293 cells and requires extended experience to deliver a batch-to-batch consistent product quality (Hernandez Bort 2019). To generally better understand and monitor cell behavior and product formation during cultivations, spectroscopic methods became more popular within the last decade (Rathore et al 2010; Mercier et al 2014; Jiang et al 2017; Wasalathanthri et al 2020). While spectroscopic methods in combination with chemometric modeling are attractive PAT tools, overlapping spectra or lack of sensitivity to particular components render some drawbacks in their broader applicability
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