Abstract
The presence of endotoxin, also known as lipopolysaccharides (LPS), as a side product appears to be a major drawback for the production of certain 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-ε-caprolactone (PCL) nanoparticles (NPs) (dP = 780 ± 285 nm) were synthesized 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 × 106 endotoxin units (EU) per mg of particle. The LPS removal efficacy increased to a higher level (~100%) 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 100% LPS removal but also ~100% protein recovery for a wide concentration range from 20–1000 μg/ml of protein solutions. The NPs were highly effective in different buffers and pHs. To scale up the process further, PCL NPs were incorporated into a supporting cellulose membrane which promoted LPS adsorption further up to ~100% just by running the LPS-containing water through the membrane under gravity. Its adsorption capacity was 2.8 × 106 mg of PCL NPs, approximately 2 -fold higher than that of NPs alone. This is the first demonstration of endotoxin separation with high protein recovery using polymer NPs and the NP-based portable filters, which provide strong adsorptive interactions for LPS removal from protein solutions. Additional features of these NPs and membranes are biocompatible (environment 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 and membrane forms, which have great potential to be employed in large-scale applications.
Highlights
In biotechnology industries, gram-negative bacteria are widely used for the production of therapeutic biomolecules including proteins, peptides, and nucleic acids[1,2,3,4,5,6]
We report first the synthesis of polymeric PCL NPs by employing a solvent evaporation method and the performances of PCL NPs for the adsorption and removal of LPS
It was found that PCL NPs in powder form removed around 88% of LPS from the water sample
Summary
Gram-negative bacteria are widely used for the production of therapeutic biomolecules including proteins, peptides, and nucleic acids[1,2,3,4,5,6]. Removing undesirable LPS from solutions is an important aim in the pharmaceutical industry and in clinical practice Conventional treatments such as coagulation and membrane filtration are adequate for removing bacteria cells and debris but not effective for removing dissolved endotoxins to a significant extent. A key step forward with the NP-based approach is to establish a high throughput, low-cost method that is not subject to high pressure-drop limitation, slow solute transport, or accompanying toxicity To this end, poly-ε-caprolactone (PCL) NPs without any modification have been manufactured in the PI’s laboratory, which are non-porous solid adsorbent nanoparticles with solute binding sites situated on the particle surface. Since PCL and cellulose are both low-cost biocompatible polymers[51,52,53], the use of such PCL NP-embedded membranes represents a novel LPS separation system that requires low capital costs but provides desirable ease of manufacturing, excellent performance, disposability, and biodegradability
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