On-Line Monitoring of Physiological Parameters of Insect Cell Cultures during the Growth and Infection Process

Abstract

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

“…Permittivity is a direct measure of the membrane enclosed volume fraction or biovolume of the cell suspension. These measurements also reflect changes in cell physiology which has been demonstrated for several cell types (Fehrenbach et al 1992;Noll and Biselli 1998;Zeiser et al 2000;Ducommun et al 2002;Cannizzaro et al 2003;Sarrafzadeh et al 2005).…”

“…However, with current sensor technology, on-line monitoring of the physiological state remains a challenging task, rendering its use as a bioprocess control parameter complex (Konstantinov 1996;Henry et al 2007). Permittivity-based in situ probes are now an established tool for the measurement of biomass in real-time in a wide variety of cell culture processes (Kamen et al 1996;Zeiser et al 1999;Elias et al 2000;Zeiser et al 2000;Ducommun et al 2001Ducommun et al , 2002Cannizzaro et al 2003). Permittivity is a direct measure of the membrane enclosed volume fraction or biovolume of the cell suspension.…”

Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

“…They are mainly related to the metabolic activity of the cell culture. 8,9 On the other hand, common direct biomass measurement methods like turbidity or in situ microscopy rather follow the total cell concentration and are insensitive to changes in the physiological state of the culture. [10][11][12] Permittivity-based biomass probes are a valuable alternative and their application in cell culture processes is rather novel.…”

“…The successful application of this technology in a wide variety of cell culture processes is described in the literature. 5,6,9,[13][14][15][16] Several studies report measurements of the permittivity at one single or two frequencies (to reduce the effect of a changing media composition on the signal). Permittivity measurements are described as a tool to directly measure the viable biomass.…”

“…21 Permittivity measurements do also reflect changes in cell physiology which has been demonstrated for a wide variety of cell types. 5,9,13,[22][23][24] Cell physiology is most commonly described by the term ''physiological state.'' This term is defined by several variables that impart significant information about the cellular state.…”