Continuous noninvasive monitoring of cell growth in disposable bioreactors

Abstract

To ensure high quality output of biotechnological processes, relevant process parameters need to be monitored. As bioprocesses are increasingly executed in single use bioreactors, there is an increasing demand for new sensors applicable to these processes. In this work, we investigate different approaches for continuous non-invasive cell growth monitoring, especially for single use bioreactor applications. Therefore, the permittivity of the cell culture is used as a measure for the biomass. In a first step, a measuring procedure based on the transmission measurement of an electromagnetic wave is investigated. It appears that the penetration depth of this sensor is not sufficient for a noninvasive measurement through the polymer wall of a single use bioreactor. Therefore, alternative setups based on magnetic induction are investigated. The initial setup is very simple. It consists of a planar coil connected to an impedance analyzer. The coil is attached to the outside of the polymer foil of the single use bioreactor and an impedance spectrum is measured. To evaluate the sensor, E. coli cultivations are performed in a modified cultivation setup, which enables measurements through the polymer foil of a Sartorius BIOSTAT® CultiBag RM, and additionally allows sampling of culture medium for optical density reference measurements. The resonance peak of the coil in the impedance spectrum, is observed as measure for the optical density. Regardless of the simple sensor construction, we found a good correlation between optical density and the damping ratio of the resonance peak. However, the sensor signal shows saturation towards high optical densities. Therefore, an LTCC coil producing a higher magnetic flux density in the culture medium is investigated subsequently. This sensor shows a linear response up to high optical densities, but the sensitivity is reduced compared to the former used coil and therefore scattering of the data is increased. However, to increase the sensitivity, a linear variable differential transformer is realized. Using this setup, the influence of the primary magnetic flux is eliminated from the measuring voltage. This approach delivers the most promising results, as the sensor response is linear up to high optical densities and data scattering is low. Fig. 1. Four-electrode measurement without (left) and with (right) polymer foil (CST Simulation at 1 kHz with discrete port excitation (1 V Amplitude), 190000 Hexahedral Mesh cells and open (add space) boundaries).

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 ].…”

Differential Inductive Sensor for Continuous Non-Invasive Cell Growth Monitoring in Disposable Bioreactors

“…Figure 6 shows a linear dependency with a coefficient of determination R 2 = 0.96 between Us' and the optical density of the culture medium up to an optical density of OD = 4. Compared to the results shown in [5], the measured sensitivity is significantly increased using the optimal measuring frequency of 108.4 kHz. …”

“…Therefore, the measuring voltage Us equals the induced voltage in Ls2. In [5], it is shown that the real part of the measuring voltage Us' depends on the polarizability of the medium (ε r ') and the imaginary part of the measuring voltage Us'' depends on the dielectric losses (εr'') and the conductivity (κ). …”

“…The limiting factor when using only a single coil is that the measuring signal is dominated by the influence of the primary flux of the coil and thus the weak secondary flux from the medium is hardly detectable. Therefore, the employment of a differential transformer for non-invasive cell growth monitoring seems to be the most promising approach [5]. In this differential setup, the influence of the primary flux through the measuring coil is eliminated from the measuring signal.…”

“…In this differential setup, the influence of the primary flux through the measuring coil is eliminated from the measuring signal. Preliminary measurements show a good correlation between the biomass concentration in the culture medium and the measuring signal [5]. However, there is no information about the influence of other parameters, like measuring frequency or filling level, on the measuring signal.…”