(126f) Effect of Fermentation Conditions on Inclusion Body Quality in High Cell Density E. Coli Fermentations

Protein therapeutics produced in high cell density cultures of Escherichia coli using industrial-scale recombinant DNA processes commonly form insoluble aggregates. These inclusion bodies are advantageous because they are often very pure aggregates of the protein of interest, and their isolation is typically a first step in purification. This study shows that fermentation conditions can influence the size, shape, and density of inclusion bodies, which may facilitate the ease of their isolation and subsequent downstream purification.

Melan-A, a promising candidate for cancer immunotherapy, was used as a model protein therapeutic that forms inclusion bodies in E. coli. Two populations of inclusion bodies appeared depending upon fermentation temperature when their density was analyzed using cesium chloride gradient ultracentrifugation. Inclusion bodies produced at 30°C were mostly about 1.18 g/ml, while the density of inclusion bodies produced at 42°C were 1.27 g/ml. At 37°C approximately 80% of the inclusion bodies were in the lower density fractions. Using electron microscopy, it was observed that the physical structure of the inclusion bodies differed. Overall, inclusion bodies produced at 30°C and 37°C were 400 nm in diameter, but the 30°C inclusion bodies had a more amorphous structure with poorly defined boundaries between subunits. Inclusion bodies produced at 42°C were 300 nm and had a relatively smooth and spherical shape. Diffusion limitations of reactions with Bradford reagent, as are expected with inclusion bodies, were exacerbated with the 42°C inclusion bodies.

Currently under investigation are the effects of additional fermentation conditions on inclusion body quality, such as the adjustment of growth rates. In addition to electron microscopy, atomic force microscopy is also being attempted to determine the mechanical properties of these inclusion bodies. Furthermore, the ability to recover a His-tagged version of the protein on a nickel column is being studied. SSX-2, another cancer antigen, and GFP are also under similar investigations to show possible generalization of the observed phenomena.