Bacterial endotoxin (lipopolysaccharide, LPS) is the main component of the cell wall of Gram-negative bacteria, which can seriously damage the immune function of the body, and further development may cause septic shock, disseminated intravascular coagulation and systemic inflammatory response syndrome. Bacterial endotoxin has a strong heat resistance and chemical stability. It can only destroy 98% by heating at 120℃ for 4 hours. To be completely inactivated, it needs to be heated at 180℃ for more than 2 hours. Endotoxin removal is quite difficult. General chemicals do not affect the activity of bacterial endotoxins, only strong acids, strong bases, or strong oxidants can destroy bacterial endotoxins. Therefore, the removal of endotoxin is important for the downstream purification of injectable drugs and biological products for the treatment of human diseases, especially genetic drugs, protein drugs, biological vaccines, and small molecule chemical drugs.
The purification methods of biological preparations mainly include various column chromatography techniques, such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), affinity chromatography (AC), molecular sieve, etc. These techniques can effectively remove endotoxin while purifying the target product. A HIC medium that can bind the target protein can be selected to remove endotoxin, because the lipid A part of endotoxin has strong hydrophobicity, but will agglutinate under high salt and cannot be hung on the hydrophobic chromatography column.
This method mainly achieves separation and purification by exchanging charged solute molecules with exchangeable ions in ion exchangers. Since endotoxin is negatively charged at pH>2 and has a strong combination with anion exchange media, it can be removed with a high-salt buffer or sodium hydroxide after elution of the target protein, which is the best way to remove endotoxin by ion exchange.
Figure 1. The principle of IEC with increasing ionic strength elution steps. (Smoluch M, et al., 2017)
Among them, especially the use of immunosorbents in production makes the purification of biomacromolecules simple. The principle of immunoadsorption is based on the specific effect of antigens and antibodies. The target protein is used as the antigen to couple the monoclonal antibody produced by hybridoma technology to the medium, and the target protein is adsorbed by the medium. The high specificity of the interaction means that only the target protein will be adsorbed on the medium, and the endotoxin will penetrate completely, resulting in a pyrogen-free and high-purity product. In spite of this, a small number of endotoxins are still adsorbable at the same time, due to the non-specific adsorption of the medium itself.
A method of separating molecules according to their size by utilizing the network structure of the gel. Typically, recombinant products have molecular weights in the thousands, while endotoxins are often polymers with molecular weights in the hundreds of thousands. Therefore, molecular sieves can effectively remove endotoxins. But its processing capacity is small and the processing time is long.
Biological agents include cell pellets, recombinant protein drugs, antibodies, vaccines, oligonucleotide drugs, etc., and their dosage forms are mostly injections, so it is very important to control their pyrogens. Once the product is contaminated with endotoxin, different measures should be selected according to the specific situation to remove endotoxin. Endotoxins are difficult to remove or inactivate due to their small size, high heat resistance, and chemical stability. The more commonly used endotoxin removal methods are mostly ultrafiltration or AC. By using AC technology, the endotoxin substrate LAL or polysaccharide B (PMB) is coupled to the gel, which is used as the medium to specifically adsorb endotoxin and allow the protein to pass through.
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