(320d) Polymeric Adsorbent for Endotoxin Removal from Biopharmaceutical Solutions

The presence of endotoxin, also known as lipopolysaccharides (LPS), as a side product appears to be a major drawback for the production of therapeutic biomolecules that are essential for research, pharmaceutical, and industrial applications. In the biotechnology industry, gram-negative bacteria (e.g., Escherichia coli) are widely used to produce recombinant products such as proteins, plasmid DNAs and vaccines. These products are contaminated with LPS, which may cause side effects when administered to animals or humans. Purification of LPS often suffers from product loss. For this reason, special attention must be paid when purifying proteins aiming a product as free as possible of LPS with high product recovery. Although there are a number of methods for removing LPS, the question about how LPS removal can be carried out in an efficient and economical way is still one of the most intriguing issues and has no satisfactory solution yet. In this work, polymeric poly-e-caprolactone (PCL) nanoparticles (NPs) (~ 780 nm) were synthesized using solvent evaporation technique at a relatively low cost and demonstrated to possess sufficient binding sites for LPS adsorption and removal with ~100% protein recovery. The PCL NPs removed greater than 90% LPS from protein solutions suspended in water using only one milligram (mg) of NPs, which was equivalent to ~ 1.5 million endotoxin units (EU) per mg of particle. The LPS removal efficacy increased to a higher level (~96%) when phosphate buffered saline (PBS containing 137 mM NaCl) was used as a protein suspending medium in place of water, reflecting positive effects of increasing ionic strength on LPS binding interactions and adsorption. The results further showed that the PCL NPs not only achieved 96 % LPS removal but also ~100% protein recovery for a wide concentration range from 10 – 1000 mg/ml of protein solutions. The NPs were highly effective in different buffers and pHs. This is the first demonstration of endotoxin separation with high protein recovery using polymer NPs which provide strong adsorptive interactions for LPS removal from protein solutions. Additional features of these NPs are biocompatible (environmental friendly), recyclable after repeated elution and adsorption with no significant changes in LPS removal efficiencies. The results indicate that PCL NPs are an effective LPS adsorbent in powder form, which have a great potential to be employed in large-scale applications.