An Advanced Monitoring Platform for Rational Design of Recombinant Processes - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 72

To overcome the limitation that dielectric spectroscopy and optical transmission
cannot deliver information on intracellular components, 2D multiwavelength
fluorescence spectroscopy has been used as a complementary monitoring system.
Therefore, the BioView spectrofluorometer (DELTA; Light & Optics, Lyngby,
Denmark), which is specially designed for online measurements [ 10 , 13 ], was
implemented in our process monitoring platform. The scan of one complete
excitation-emission matrix (150 excitation-emission wavelength pairs) takes
about 90 s, which enables almost continuous measurement. Within a cell, fluo-
rescent components such as tyrosine, tryptophan, riboflavin, and pyridoxine are
present, and their fluorescence depends to a certain degree on the cellular state.
Although wavelength combinations can be associated with these fluorescent
compounds, direct calibration of fluorescence signals versus process variables was
not possible [ 38 ].
2.3 Monitoring of Products from the Recombinant System
When recombinant cellular systems are used, quantification and monitoring of
expressed products are of high priority. In particular, there are effects triggered by
the recombinant system which are significant. Hence, a target-oriented offline
monitoring approach must aim at qualification and quantification of recombinant
components and simultaneously allow for measurement of the dynamics of the
cellular responses triggered by the recombinant gene expression.
One-dimensional sodium dodecyl sulfate (SDS) gel technology is often employed
for semiquantitative product analysis, for determination of the solubility distribution
(ratio of soluble to insoluble recombinant protein), and in combination with Western
blotting, for identification of target proteins and fragments thereof [ 43 ].
Enzyme-linked immunosorbent assay (ELISA) technology was used for
quantification of green fluorescent protein (GFP) and human superoxide dismutase
(SOD), two model proteins used to evaluate the platform. Protein-specific prop-
erties such as the autofluorescence of GFP or the enzyme activity of SOD can also
be exploited to gain information on protein quantity and quality [ 44 - 46 ].
Formation of isoforms of the recombinant target proteins, e.g., due to addition of
acetyl residues or phosphoryl groups or false processing during synthesis, is
another important aspect of protein quality. To obtain this information, product
samples are separated by narrow-range 2D electrophoresis gels and analyzed by
MS after excision and digestion. Figure 1 shows isoforms of SOD produced with
E. coli HMS174(DE3)(pET11aSOD) [ 47 , 48 ].
The plasmid copy number representing the gene dosage is calculated from
plasmid and chromosomal DNA according to Breuer et al. [ 49 ].
Real-time PCR (RT-PCR) is used to quantify messenger RNAs (mRNAs) for
recombinant product genes and T7 RNA polymerase [ 50 ].