Process Analytical Technology (PAT) in Insect and Mammalian Cell Culture Processes: Dielectric Spectroscopy and Focused Beam Reflectance Measurement (FBRM)

Abstract

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells

“…Optimal timing guided by process analytical technology is necessary during scale up Appropriate process analytical technology is required during scale-up to monitor the physiological state of the cells, which is a critical process parameter for protein production (Junker et al 1994). Dielectric spectroscopy (Druzinec et al 2013(Druzinec et al , 2014Justice et al 2011) and online OD measurement (Akhnoukh et al 1996;Bédard et al 1994) are valuable tools for the real-time monitoring of processes based on insect cells, such as the baculovirus mediated protein expression in Spodoptera frugiperda cells. Exemplarily, in a corresponding process for recombinant adenoassociated virus vectors, the permittivity signal was used to determine the optimal time of infection and harvest (Negrete et al 2007).…”

Dielectric Spectroscopy and Optical Density Measurement for the Online Monitoring and Control of Recombinant Protein Production in Stably Transformed Drosophila melanogaster S2 Cells

“…Biomass can be quantified indirectly by off-gas analysis to measure respiration [ 2 , 3 ], 2D fluorescence spectroscopy to calculate the NAD(P)H content [ 4 , 5 ], biocalorimetry to monitor metabolic heat [ 6 ], or a combination of process data using soft sensors [ 7 ]. Direct methods include cell counting by in situ microscopy [ 8 ], near-infrared (NIR) spectroscopy [ 9 ], online optical density measurements [ 3 , 10 , 11 , 12 ] and dielectric spectroscopy [ 6 , 13 , 14 , 15 ]. Regardless of the chosen strategy, online biomass monitoring systems must meet several requirements [ 16 ].…”

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

“…Alternatively, an intercalating fluorescent dye such as SybrGreen I can be used to estimate DNA levels, which are linearly related to the cell density, and this can be achieved without cell detachment [86]. Dielectric spectroscopy is a promising tool for the online measurement of cell density [21,86,87]. At a frequency of 300 kHz, there is a linear correlation between the permittivity and the cell density up to 5 × 10 4 cells/cm 2 (∼80% confluence).…”