Influence of the hydromechanical stress and temperature on growth and antibody fragment production with Bacillus megaterium

Abstract

Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the temperature and the hydromechanical stress play a very important role for significant product formation during process development or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs of about 0.5 kW/m(3) (agitation rates around 500 min(-1)) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input of about 0.06 kW/m(3) (agitation rate around 250 min(-1)) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew at low volumetric power inputs and high cultivation temperatures.

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

“…Especially the EV was more than halved when the culture developed from exponential to stationary phase. Therefore, normalization by EV values at calculating fluorescent concentration (FL-FC) is very important to interpret fluorescence data sets [ 30 ]. Region analysis of dot plots (FL1-FC vs. FL3-FC) was done and percentages of cells in particular regions were determined for all sample points (Figure 8A ).…”

“…for every counted particle no matter if this is a single bacterium or a chain of bacteria. Normalizing fluorescence to cell size is done per event, so that the normalization actually represents fluorescence per cell or cell conglomerate no matter how many (single or chains) or how big these cells (single or chains) are [ 30 ].…”

Expression of Recombinant Antibodies

“…The Gram-positive bacteria Bacillus brevis (76, 77), Bacillus subtilis (78, 79), and Bacillus megaterium (80–85) have already been successfully used for the production of different antibody fragments. In addition, B. megaterium does not produce alkaline proteases and provides high stability of plasmid vectors during growth allowing stable transgene expression during long term cultivation in bioreactors (86).…”

Influence of fructose and oxygen gradients on fed‐batch recombinant protein production using Bacillus megaterium

“…By normalizing fluorescence to cell size per event, fluorescence is given per cell or cell conglomerate no matter how many (single or chains) or how big these cells (single or chains) are [26]. By normalizing the values from FL1 by the so-called electronic cell volume (EV -Coulter Counter Principle, Cell Lab QuantaTM SC MPL), a single-cell fluorescence concentration (FL1-FC) could be estimated.…”

“…for every counted particle no matter whether this is a single bacterium or a chain of bacteria. By normalizing fluorescence to cell size per event, fluorescence is given per cell or cell conglomerate no matter how many (single or chains) or how big these cells (single or chains) are [26]. By these means, shifts in fluorescence intensity of GFP producing cells could be determined for every sample by calculating the mean values of the FL1-FC distributions.…”