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.