Software sensors for fermentation processes.

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R(O/S) sensor which is the ratio between oxygen and energy substrate consumption. The R(O/S) variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R(O/S) sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry.

Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes.

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml(-1)) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml(-1) dropped to approximately 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml(-1) probably reflects a loss of cell division capability rather than cell death.