Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed-batch fermentation processes using multiparameter flow cytometry

Abstract

Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti-lysozyme Fab fragment under the control of the lac-based expression system, were taken at various stages from fed-batch fermentation processes and stained with a mixture of bis-(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration were made. Depending on time of induction the maximum amount of Fab accumulating in the supernatant varied quite markedly from 1 to 4 microg ml(-1) as did subsequent cell physiological state with respect to PI/BOX staining with a concomitant drop in maximum biomass concentration. Depending on point of induction a fourfold increase in Fab production could be achieved accompanied by a approximately 50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry.