Biomedical Engineering Reference
In-Depth Information
Recombinant protein production exposes cells to high stress levels and can
cause cell death in the course of the production process. As the employed NIR
technology does not give any information on cell viability, dielectric spectroscopy
was selected as an alternative. This technique makes use of the electrical properties
of cells exposed to a radiofrequency electrical field. The nonconducting nature of
the cell plasma membrane allows a buildup of charge, so that cells with intact
plasma membranes act as capacitors. The resulting capacitance is proportional to
the membrane enclosed volume and thereby to the concentration of viable cells
[ 36 , 37 ]. The ABER biomass monitor BM214 M allowing for capacity measure-
ments at two frequencies is employed (Aber Instruments Ltd., Aberystwyth, UK).
Although the capacity signal shows a strong correlation with the cell dry mass
(CDM), a direct calibration of capacity versus CDM is not feasible as there are
changes in cell size in the course of the process [ 38 ].
Flow cytometry is used (i) to gain offline information on total cell number
(TCN) via ratiometric counting and (ii) to determine the percentage of dead cells
(DC) by discrimination of dead cells via staining with propidium [ 38 ].
In addition to this set of methods, additional information on cell growth is
derived from measurement of base consumption during the process. During growth
of bacteria, acids are produced, and there is a close correlation of cell growth with
release of acidic metabolites and thereby also with the amount of base needed to
maintain the pH at a constant level. In case of using NH 4 OH for pH control, for
each uptake of NH 4 + a proton is excreted, which in turn triggers the addition of
NH 4 OH. By considering the buffer capacity of the nutrient medium, a close cor-
relation between NH 4 OH consumption and growth can be derived.
2.2 Monitoring of Metabolites and Cell Compounds
The composition of the extracellular medium changes continuously during the
process.
There
are
decreasing
concentrations
of
media
compounds
but
also
changes in the concentrations of cellular metabolites secreted by the cells.
HPLC methods (offline mode) are used to quantify concentrations of sugars,
amino acids, and metabolites such as acetate and ethanol in the fermentation broth
medium [ 39 ].
In addition, a method for online measurement of volatile organic compounds
(VOC) is implemented [ 40 ]. The commercially available method of proton transfer
reaction mass spectrometry (PTR-MS; Ionicon Analytik GmbH, Innsbruck, Aus-
tria) [ 41 , 42 ] is a highly sensitive chemical ionization technique that enables soft
ionization without fractionation and measurements of individual VOCs ranging
from pptV (10 -12 [vol/vol]) to ppmV (10 -6 [vol/vol]) levels. VOCs are ionized via
proton transfer reactions from H 3 O + ions and are detected by MS at 1 atomic mass
unit (amu) higher (M +1 ) than the relative molecular weight (M r ) of the neutral
compounds. This device allows real-time VOC quantification and noninvasive
sampling without any sample preparation steps.
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