Biomedical Engineering Reference
In-Depth Information
off-line methodology is generally required prior to validation of the in-line technology.
Sometimes, differences in the measuring technology off-line and in-line will not permit
direct comparison. Model revalidation is necessary when changes to the processes occur
that could influence the measurement device or the analyte of interest.
The OD probe has been used successfully to monitor biomass in fermentation
reactors and attempts have been made to use it tomeasure biomass in cell culture but with
significant obstacles. OD probes are useful in determining biomass in fermenters
because of the high viable cell density and brief duration of most fermentation processes.
Wu et al. [19] have tested several optical density probes and determined that a
transmittance probe is the type best suited for determining total cell density (TCD) in
mammalian cell culture. Since manymammalian cell culture processes run to lower than
70% cell viability, interfering absorbance from dead cells and cell debris may overesti-
mate the actual viable cell density. In a head-to-head comparison of an optical density
probe and a capacitance probe, Schmid and Zacher [20] determined that at lower cell
densities, turbidity interferes with the ability to measure biomass accurately. For cell
culture processes, optical density probes are better suited for applications where viable
cell densities are maintained at high levels, such as during a seed train expansion step. If
properly implemented, they should be able to provide timing and volume of seed reactors
to subsequent seed bioreactor or to the production reactors. If the OD reading is slightly
out of tolerance, adjustments can be made to the volume delivered to maintain the
consistency of the total cell density transferred. To make the technique usable in cell
culture, regression coefficients obtained from the calibration cure are used to predict
TCD on the basis of OD readings.
Capacitance probes, or dielectric spectroscopy probes, are the other type of probes
used to assess viability in mammalian cell culture. The benefit of a capacitance probe is
that it measures capacitance of intact cell membranes, so only product-producing cells
are measured. In addition, capacitance probes are less sensitive to dead cells, bubbles,
and dissolved solids than optical density probes since they do not rely on light scatter. It
has been clearly demonstrated that these in-line probes yield readings that tightly
correlate with off-line viable cell densities by automated hemacytometer or other such
devices (Fig. 12.2).
One concern with capacitance probes is their ability to give accurate readings with
cell cultures that have different cell morphologies within the same culture. While the
permittivity at a given frequency is linearly related to viable cell density (biovolume),
some probes have been designed with parallel multifrequency measurements (dielectric
spectroscopy) to access the information on different physiological and morphological
cell phases. The very limited ionic permeability of the plasma membrane gives living
cells specific dielectric properties. At high frequencies, above 10MHz, the ion move-
ment amplitude is limited and is not significantly affected by the presence of
the cytoplasmic membrane. A cell suspension has nearly the same overall electric
properties as the suspendingmedium. At lower frequencies, around the critical frequency
(fc), the displacement amplitude is higher, ions accumulate at the membrane, and the
cells behave as tiny capacitors. The global charge accumulation depends on the viable
cell density. When measured at a fixed frequency close to the average fc, the permittivity
is independent of cell size and state. At very low frequencies, when the cells are fully
