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), OD 600nm 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 mg ml 21 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 $50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry. ' 2008 International Society for Advancement of Cytometry

Assessment of the manufacturability of Escherichia coli high cell density fermentations

“…This indicates that the use of the same disruption intensity can be used to measure cell strength which declines as the culture is subjected to longer bioreactor operation. This is in accordance with Want et al 31 who noted a progressive and detrimental change in cell physiological state (also using E. coli W3110) with respect to permeability using flow cytometry. From a processing perspective, this leads to weaker host cell outer membranes that have the potential to damage on entering the high shear zones of industrial centrifuges during the subsequent solid-liquid separation.…”

“…35 Also, the increase in the amount of product in the periplasmic space with culture age may apply pressure on the outer cell membrane resulting in outer membrane weakening. 31 Micronization susceptibility has important implications for unit operations further downstream both at a technical and economic level. Smaller cell debris that cannot be efficiently well separated during solid-liquid separation will create unacceptable fouling of clarifying filters that would need to be of a bigger size (larger filtration area) to avoid a decrease in clarifying efficiency.…”

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

“…Sundstöm et al [60] studied the effects of inclusion body formation on cell physiology during the production of promegapoietin by E. coli. Similar observations were made using the same microbial chassis but with another recombinant protein (Fab antibody fragment) leading to the formation of inclusion bodies [63]. In related studies, it was demonstrated that the microbial chassis influences the heterogeneity of recombinant protein production.…”

Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures

“…In both cases within 4 h of inoculation almost all cells were either only Chamsartra et al, 2005;Amanullah et al, 2002) and the dual staining combination with PI/bisoxanol is suitable for Gram -ve cells e.g. E. coli, Salmonella spp., Acinetobacter johnsonii (Boswell et al, 1998;Want et al, 2009.;Hewitt & Nebe-von-Caron 2001;Nebe-von-Caron et al, 2000;Boswell et al, 1998). This is likely to be species and process specific whilst the interpretation of such data can only be reliably …”