Automated Measurement and Monitoring of Bioprocesses: Key Elements of the M3C Strategy - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 17

population is, e.g., highly active, what percentage is dormant but functional, and
what percentage is dead. Viability probes can be selected from a wide variety of
commercially available fluorescent dyes, depending on the instrument equipment
available (lasers, filters, and detectors) and the focus of the analysis, for instance,
membrane integrity, intracellular enzymatic activity, uptake of substrates, or
conversion of substrates [ 60 , 61 ]. Part of the dyes is membrane permeant and
another part is membrane impermeant, which can be exploited for differentiation.
Multiple staining could be used to monitor cell physiological responses of bacterial
batch cultures and provided important physiological information at the single cell
level [ 62 ].
An alternative is the exploitation of fluorescent reporter molecules produced by
the cells, e.g., green fluorescent protein (GFP). For instance, this can be linked to a
protein of interest and used to indirectly monitor the cell-specific production of the
target product, as long as this is associated with the cells (intracellular or peri-
plasmic). The method may involve a fusion protein [ 51 ] or linkage by an internal
ribosome entry site [ 63 ]. Using online FCM, Broger et al. [ 58 ] found that, in a
Pichia pastoris cultivation, only 75 % of the cells growing in the population were
able to produce, with widely varying titers, whereas 25 % of all cells were absolute
nonproducers. Such distinction at the single cell level and thus quantitative
characterization of the performance of the production strain cannot be acquired
with any other technique established to analyze products in a population-averaging
manner (e.g., gel electrophoresis, blotting, ELISA, chromatography, mass spec-
trometry, etc.) nor with in situ online fluorescence sensors.
Cell cycle and synchronization studies are predestined to use FCM (e.g., [ 64 ,
65 ]): a great variety of nucleic acid (both DNA and RNA) dyes are readily
available nowadays. They also permit quite sensitive analysis for contaminants
accumulating in a bioprocess [ 66 ], specifically because several tens or hundreds of
thousands of events can be screened in a short time.
Besides fluorescence events, FCM also detects light scattering events. The
small-angle forward-scattered light is linked to the size of a particle, whereas side-
scattered light is linked to the granularity or intracellular structure of a cell. Both
characteristic properties may undergo significant changes during a bioprocess.
A review of recent developments, including online applications of FCM, is given
by Diaz et al. [ 67 ], highlighting features such as obtaining population distribution
information at the single cell level, validating more accurate kinetic models, and
using the data for process control and optimization.
3.2.6 Sizing of Particles and Solutes
After a decade or so of dormancy, field flow fractionation (FFF) techniques and
apparatuses recently appeared on the market. However, FFF is mostly used in lab-
oratories, not online during processes, although this may only be a matter of time.
FFF is an elution/separation technique suitable for molecules with molecular weight
of approximately [1,000 Da up to particle size of some 100 lm. A separating