Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations.

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 µg/mL compared with ca. 118 and 990 µg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain.

Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.