The in‐line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology

Abstract

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.

Scaled‐up manufacturing of recombinant antibodies produced by plant cells in a 200‐L orbitally‐shaken disposable bioreactor

“…The MTAD#31.2 cells exhibited a shorter lag phase and therefore faster growth compared to previous reports describing BY-2 growth parameters (Holland et al, 2010;Vasilev et al, 2013), which could be attributed to differences in the medium composition and/or adaption of the cells to the medium. The doubling times of 22.3-23.5 h were similar to those previously described for non-transgenic (Nagata and Nemoto, 1992) and transgenic BY-2 cells (Havenith et al, 2014;Holland et al, 2013). In conclusion, our data prove that the orbitally-shaken SB200-X bioreactor is an efficient and highly scalable platform for the production of biopharmaceutical proteins using tobacco BY-2 cells.…”

“…Several types of stainless steel or disposable bioreactor systems have been used for the cultivation of plant cells, including stirred‐tank bioreactors (Holland et al, ; Huang, ; Reuter et al, ; Schmale et al, ; Trexler et al, ), bubble column reactors (Huang et al, ), single‐use bubble column bioreactors (Shaaltiel et al, ), wave‐mixed bioreactors (Eibl and Eibl, ; Huang and McDonald, ; Raven et al, ), and miniature bioreactors for high‐throughput screening (Eibl et al, ; Huang and McDonald, ). Among those systems, wave‐mixed bioreactors have been widely used for the cultivation of hairy roots and plant cells that benefit from constant illumination, such as mosses (Eibl and Eibl, ; Weathers et al, ).…”

Plant cell culture strategies for the production of natural products

“…In this context, promising new strategies are emerging that hold significant promise for future application. For example, the use of a non-invasive, in-line system to monitor cell biomass during culture can be an important step to maximize product yield and quality, while improving culture nutrients (36) . Also, culture parameters including levels of phytohormones and components within the media can be optimized more rapidly by employing a statistical design of experiments approach (37) .…”

Putting the Spotlight Back on Plant Suspension Cultures

“…Plant cells are 50–200 μM in length and range in morphology from spherical to cylindrical depending on the growth phase. Cells in the exponential growth phase undergoing rapid division are spherical or elliptical, with a length of 50–100 μm, whereas those at the end of the exponential growth phase grow mainly by elongation and tend to be more cylindrical, with a length of up to 200 μm (Mavituna and Park, 1987 ; Holland et al, 2013 ). Aggregation occurs when daughter cells fail to separate after cell division, and is promoted by extracellular polysaccharides.…”

“…Although RFIS measures the volume of viable cells, this parameter correlates well with the packed cell volume, wet cell weight, and dry biomass weight. Continuous measurement can also pinpoint the transition from cell division to cell elongation (Holland et al, 2013 ).…”