Establishment of a scalable seed train is an integral part of any fermentation process development initiative. Multi-stage seed trains use a combination of shake flasks and bioreactors to expand the culture biomass to reach proper inoculum densities for the production fermentor.  Generally, for bacterial systems, there is little difference between the growth in shake flasks and bioreactors, but a recent study at MedImmune has shown otherwise.  As part of MedImmune's development of a fermentation process using recombinant Escherichia coli HMS174, we grew the second stage of a two-stage seed train in a bioreactor under high agitation with an inoculation level of 0.1% from a mid-exponential phase shake flask culture using a complex medium.  Under these conditions, we have observed no growth at three different fermentor scales (1-L, 15-L and 100-L), whereas parallel shake flask cultures under the same conditions grow at acceptable rates.  Upon further investigation, we have found that E. coli HMS174 is sensitive to a combination of agitation, cell density (inoculation level), and, to a lesser degree, antifoam concentration.  At the 1-L scale, agitation rates of < 400 rpm have supported growth with a 0.1% inoculation level.  Further investigation has shown that growth is supported at agitation rates of up to 1200 rpm but with a 1.25% inoculation level. Additionally we tested the growth of null and recombinant versions of both E. coli BL21(DE3) and DH5a cells under a subset of the experimental conditions.  We found that BL21(DE3) grows well at high agitation rates and low inoculation levels, while DH5a is more sensitive to agitation at low inoculation levels.  Based on these small-scale findings, our final production seed train for the recombinant E. coli HMS174 strain incorporates considerable modifications to agitation rate, inoculation level, and antifoam concentration to achieve an acceptable growth profile.

Sensitivity of null and recombinant <i>Escherichia coli</i> HMS174 to high agitation rates and low inoculation levels:  A cautionary tale for seed train design

243rd National Meeting (2012)

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Sensitivity of null and recombinant Escherichia coli HMS174 to high agitation rates and low inoculation levels: A cautionary tale for seed train design