Observer‐based online compensation of inner filter effect in monitoring fluorescence of GFP‐expressing plant cell cultures

Abstract

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

“…Currently, process monitoring for a plant cell culture process is mainly applied for detecting cell growth and related physiological status such as biomass concentration, nutrient consumption, OUR, carbon dioxide production rate (CPR), respiratory quotient (RQ), DO and pH. Approaches have been proposed for off-line or on-line estimation of cell concentration in plant cell cultures based on the changes on medium conductivity [157], osmolarity [158], dielectric properties [159,160], and culture turbidity [161,162]. McDonald et al [163] applied a focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA) to accurately characterize several plant suspension cultures (Oryza sativa, Nicotiana benthamiana and Trichosanthes kirilowii) including biomass concentration and aggregate size.…”

“…To rapidly observe the product production kinetics and cellular dynamic responses to recombinant protein production, the recombinant protein can be fused with GFP, allowing on-line monitoring of the recombinant protein production by measuring the GFP fluorescence in the cell culture [166]. Furthermore, Su et al [161] recently applied the extended Kalman filter and on-line measurement of GFP fluorescence to develop a model-based state observer, allowing accurately estimation of GFP concentration in cell culture. Studies using the extended Kalman filter [167] and artificial neural network [168] have developed software sensors for monitoring and estimating biomass concentration in plant cell culture.…”

Biomass measurement online: the performance of in situ measurements and software sensors

“…Excitation wavelengths are usually around 250-550 nm and emission wavelengths around 300-600 nm. Subtraction spectra are required when process conditions vary during cultivations, but this is often compensated in commercial software that accompanies products [57]. 2D spectroscopy has been applied to Saccharomyces cerevisiae [24,39,59], Escherichia coli [39,59], Claviceps purpurea [10,39], and Sphingomonas yanoikuyae [39].…”

A method for determining supernatant-free spectra generated from fluorescently stained cells in suspension

“…An often used approach for the determination of the content and/or spectral properties of the cell-bound fluorochromes is based on collecting the cells by centrifugation and recording the fluorescence from the supernatant, which is then subtracted from the total fluorescence of the cell suspension; see for example [6][7][8]. Though this approach seems to be feasible at first glance, it bears a problem caused by the scattering of both the excitation and emission light, which can significantly affect the intensity of fluorescence measured in turbid cell suspensions [9][10][11]. In that case, therefore, the simple subtraction of the supernatant fluorescence from the total fluorescence will yield both erroneous intensities and distorted spectra of the bound-dye emission.…”