Combined bioreaction and separation in a simulated counter-current chromatographic bioreactor-separator system

The objective of this work has been to investigate the principle of combined bioreaction and separation in a simulated counter-current chromatographic bioreactor-separator system (SCCR-S). The SCCR-S system consisted of twelve 5.4cm i.d x 75cm long columns packed with calcium charged cross-linked polystyrene resin. Three bioreactions, namely the saccharification of modified starch to maltose and dextrin using the enzyme maltogenase, the hydrolysis of lactose to galactose and glucose in the presence of the enzyme lactase and the biosynthesis of dextran from sucrose using the enzyme dextransucrase. Combined bioreaction and separation has been successfully carried out in the SCCR-S system for the saccharification of modified starch to maltose and dextrin. The effects of the operating parameters (switch time, eluent flowrate, feed concentration and enzyme activity) on the performance of the SCCR-S system were investigated. By using an eluent of dilute enzyme solution, starch conversions of up to 60% were achieved using lower amounts of enzyme than the theoretical amount required by a conventional bioreactor to produce the same amount of maltose over the same time period. Comparing the SCCR-S system to a continuous annular chromatograph (CRAC) for the saccharification of modified starch showed that the SCCR-S system required only 34.6-47.3% of the amount of enzyme required by the CRAC. The SCCR-S system was operated in the batch and continuous modes as a bioreactor-separator for the hydrolysis of lactose to galactose and glucose. By operating the system in the continuous mode, the operating parameters were further investigated. During these experiments the eluent was deionised water and the enzyme was introduced into the system through the same port as the feed. The galactose produced was retarded and moved with the stationary phase to be purge as the galactose rich product (GalRP) while the glucose moved with the mobile phase and was collected as the glucose rich product (GRP). By operating at up to 30%w/v lactose feed concentrations, complete conversions were achieved using only 48% of the theoretical amount of enzyme required by a conventional bioreactor to hydrolyse the same amount of glucose over the same time period. The main operating parameters affecting the performance of the SCCR-S system operating in the batch mode were investigated and the results compared to those of the continuous operation of the SCCR-S system. . During the biosynthesis of dextran in the SCCR-S system, a method of on-line regeneration of the resin was required to operate the system continuously. Complete conversion was achieved at sucrose feed concentrations of 5%w/v with fructose rich. products (FRP) of up to 100% obtained. The dextran rich products were contaninated by small amounts of glucose and levan formed during the bioreaction. Mathematical modelling and computer simulation of the SCCR-S. system operating in the continuous mode for the hydrolysis of lactose has been carried out. .

Setting up a bioreactor for recombinant protein production in yeast

Scale-up from shake flasks to bioreactors allows for the more reproducible, high-yielding production of recombinant proteins in yeast. The ability to control growth conditions through real-time monitoring facilitates further optimization of the process. The setup of a 3-L stirred-tank bioreactor for such an application is described. © 2012 Springer Science+business Media, LLC.

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 105 cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity. © 2013 Springer Science+Business Media Dordrecht.

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.