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

Abstract

Lab and pilot scale batch cultivations of a CHO K1/dhfr À host cell line were conducted to evaluate on-line multifrequency permittivity measurements as a process monitoring tool. The b-dispersion parameters such as the characteristic frequency (f C ) and the permittivity increment (De max ) were calculated on-line from the permittivity spectra. The dual-frequency permittivity signal correlated well with the off-line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off-line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C M ), and effects caused by medium conductivity (r m ) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f C as a result of a fall in intracellular conductivity (r i ) was identified as responsible for the observed changes in the dual-frequency permittivity signal. It is hypothesized that the b-dispersion parameter f C is indicative of changes in nutrient availability that have an impact on intracellular conductivity r i . On-line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control.

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

“…Adding extra nutrient resulted in a less prominent change of the on-line signal. We observed significant changes in cell diameter during phase FB I, similar to those observed at the start of the cultivation for previously reported batch experiments (Ansorge et al 2010) (Fig. 1c).…”

“…Although the second feed addition should have resulted in a change in the nutrient availability there was no direct change in f C . Thus, f C was affected by changes in nutrient state as well as cell size which is in-line with the underlying theory (Schwan 1957;Harris et al 1987;Ansorge et al 2010). This became even more evident in phase FB III when cell size remained almost constant for the entire cultivation phase and the first part of phase FB IV.…”

“…First, a CHO K1/ dhfr -(dehydrofolate reductase negative) host cell line was cultivated in fed-batch mode (Ansorge et al 2010). …”

“…Multi-frequency permittivity measurements can be used to monitor nutrient limitations during batch cultivations of CHO cells (Ansorge et al 2010). In this previous study, a decrease in the on-line dual frequency permittivity signal (De Fogale ) was an indicator for a metabolic shift after glutamine depletion.…”

Process control in cell culture technology using dielectric spectroscopy

“…This was confirmed for other cell lines and systems [Ansorge et al, 2007]. In 2010, similar results were published for a batch process of CHO cells on the laboratory and pilot scale [Ansorge et al, 2010].…”