Fluorescence‐based soft sensor for at situ monitoring of chinese hamster ovary cell cultures

Abstract

Multi-wavelength fluorescence spectroscopy was investigated as a potential tool for use in monitoring key process variables that include: viable and dead cells, recombinant protein, glucose, and ammonia concentrations for Chinese hamster ovary (CHO) cells during cultivation.For the purpose of calibrating the fluorescence-based empirical model, cells were grown in batch mode with different initial glucose and glutamine concentrations.Spectrofluorometer settings were optimized to ensure reproducibility and accuracy of the acquired spectra. With the purpose of gaining qualitative insight into the evolution of the spectra, the trajectories of individual fluorophore peaks were studied during the cultivation process. Spectral changes related to biomass and secreted proteins were investigated by comparing the spectra at various stages during the downstream processing. A partial least square regression (PLSR) was used to formulate empirical models that related the input data set, i.e., the fluorescence excitation-emission matrix, to the actual state of the system including viable cell and dead cells and recombinant protein, glucose, and ammonia concentrations. The models exhibited accurate prediction ability for the process variables of interest.

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno‐associated virus production by fluorescence spectroscopy

“…Examples include prediction of biomass and recombinant protein production in Escherichia coli (Schwab et al, ) and prediction of product titer, glucose consumption and optical density in yeast cultures (Zabadaj, Chreptowicz, Mierzejewska, & Ciosek, ), to name a few. Our group and others have also shown the applicability of fluorescence spectroscopy to monitor viable cell concentration and recombinant protein titers in mammalian cell culture systems (Ohadi et al, , , Teixeira et al, , ), or critical quality attributes such as monoclonal antibody aggregation (Schwab & Hesse, ).…”

“…Several spectroscopic techniques have been used for cell culture process and virus production monitoring, with Raman (Rangan et al, ; Santos, Kessler, Salou, Menezes, & Peinado, ; Webster, Hadley, Hilliard, Jaques, & Mason, ), near‐infrared (Mercier et al, ; Rowland‐Jones, van den Berg, Racher, Martin, & Jaques, ), dielectric (Kroll, Stelzer, & Herwig, ; Mercier et al, ; Nikolay, Léon, Schwamborn, Genzel, & Reichl, ; Petiot, Ansorge, Rosa‐Calatrava, & Kamen, ) and fluorescence spectroscopy (Karakach et al, ; Schwab & Hesse, ) being the most widely used. All possess characteristics desirable for PAT: spectroscopic techniques are noninvasive, nondestructive, and able to provide rapid information from several components simultaneously (Ohadi, Aghamohseni, Legge, & Budman, ; Ohadi, Legge, & Budman, ; Rowland‐Jones et al, ; Teixeira et al, , ), including product quality attributes (Chopda, Pathak, Batra, Gomes, & Rathore, ; Li et al, ). Both Raman and fluorescence have proven worthy for monitoring recombinant protein production and cell density in cell culture processes, due to their ability to track metabolite dynamics in the complex cell culture media of mammalian cells (reviewed in Abu‐Absi et al, ).…”

“…Fluorescence spectroscopy has the advantages of a more straightforward spectra acquisition, preprocessing and interpretation, coupled with high sensitivity and low cost. It also has the ability to detect fluorophores inside and outside the cells (Ohadi et al, ; Schwab, Lauber, & Hesse, ; Teixeira et al, , ; Wolf et al, ). When compared with dielectric spectroscopy for monitoring virus and protein production, fluorescence has the advantage of incorporating information from metabolite dynamics, rather than using changes in cell physiology (Negrete, Esteban, & Kotin, ; Petiot et al, ; Zeiser, Elias, Voyer, Jardin, & Kamen, ).…”

Generic Raman‐based calibration models enabling real‐time monitoring of cell culture bioreactors

“…Furthermore, other spectroscopy-based methods such as such as NIR, MIR, and fluorescence spectroscopy have been used to monitor CHO culture process parameters. [22][23][24] Results from preliminary studies indicate Raman is an excellent candidate for process monitoring and inline measurement of cell culture process parameters. In this study, we build upon previous work to generate generic calibration models using a comprehensive data pool from several mammalian cell culture batches.…”

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

“…Among the available spectroscopic techniques [6][7][8], 2D fluorescence spectroscopy (2DFS) proved to be a reliable method which allowed online monitoring of viable cell concentrations as well as product and metabolite concentrations in mammalian cell batch and fed-batch cultivations with non-fluorescent feed [9]. Ohadi et al (2014) described that it is possible to predict key process parameters at-line in Chinese hamster ovary cell (CHO) shake flask cultures [10]. Schwab et al (2016) proved that it was possible to predict fluorescent and non-fluorescent key process parameters online in CHO fed-batch cultivations with highly fluorescent feed medium.…”