Techniques for Downstream Removal of Endotoxins
Downstream process for pharmaceutical manufacturing comprises of three steps: (1) initial recovery by extraction or isolation, (2) purification and (3) polishing 1-3. Endotoxin removal presents a unique challenge which form stable interactions with themselves and possibly with target therapeutics.
UltrafiltrationA single endotoxin molecule in its monomeric form has a molecular weight 10-30 kDa 22 depending on the core polysaccharides and oligosaccharide chain, but endotoxins have the ability to aggregate and form micelles and vesicles with molecular weights above 1000 kDa 29 and diameters up to 0.1 μm 20. The endotoxin micelles and vesicles can be separated from water, salts, and small target therapeutic molecules through size exclusion in ultrafiltration. Factors that affect the removal of endotoxins from aqueous solutions include the size distribution of the molecules in solution, the interactions between target molecules and endotoxin, therapeutic protein concentration and the presence of detergents. The effect of protein concentration on the endotoxin removal efficiency using ultrafiltration membranes has been explored 22,110. Ultrafiltration membranes with 100 kDa molecular weight cut off (MWCO) were used to filter endotoxin contaminated protein solutions with concentrations varying between 2-30 mg/ml. The % endotoxin removal through the membrane ranged from 28.9% to 99.8%, depending on the level of protein concentration and endotoxin dilution. The more dilute the protein samples were made the higher was the rate of removal. A possible explanation for such removal efficacy is the reduction of endotoxins with each protein dilution step and the formation of endotoxin monomers compared to micelles and vesicles in dilute solutions.
Effects of detergent concentrations on the interactions between endotoxin molecules was studied and contribute towards efficient endotoxin removal. Multiple Tween 20 of 0.0%, 0.5%, 1.0%, and 2.0% were added to the protein solutions and then the respective removal efficiency was calculated 22. An increase in the Tween 20 concentration led to an increase in the passage of endotoxin into the permeate and thus removing endotoxin from protein 22. These results demonstrate that the presence of a detergents decreases the size distribution of endotoxin aggregates. As the detergent concentration increased, the equilibrium shifted from micelles and vesicles to monomers 22,110. This method is undesirable for ultrafiltration where endotoxin monomers are to be trapped within the membrane and desired protein be allowed to pass as they are less likely to be stopped by the filtration membrane compared to endotoxin aggregates.
Ultrafiltration has been used to separate endotoxin molecules from small target therapeutic drug molecules. For example, ultrafiltration was utilized to separate endotoxin aggregates from BMS-753493, a small aqueous drug molecule with a molecular weight of 1.57 kDa 111. Two membrane sizes were used to perform endotoxin decontamination of the drug molecules: 3 kDa and 10 kDa. Both ultrafiltration membranes were effective in reducing the endotoxin concentration to below 0.03 EU/mg but compared to the MWCO of 3 kDa, the 10 NMWCO had higher drug yield of around 95% unlike the 3 kDa membrane which lost around 55% of the desired product 111 . Thus, ultrafiltration membranes are an effective tool for removing endotoxins from aqueous drug molecules and other therapeutic products 111.
The main limitation associated with the ultrafiltration technique is that in most cases it can be used to remove endotoxins from molecules that are magnitudes smaller than the endotoxin aggregates.  For this reason, this method is not applicable for most endotoxin separation scenarios. Ultrafiltration is best suited for removing endotoxin from water, salts, or small molecule therapeutics that do not have an affinity for endotoxin.