Endotoxin is lipopolysaccharide(LPS) derived from the outer membrane of the cell wall of Gram-negative bacteria. It consists of lipid A, core polysaccharide (C-OS) and O-specific polysaccharide(O-PS). Endotoxin is a major pyrogen substance in biological products, even a minor amount of which can cause inflammation, fever, vascular thrombosis, organ failure, shock and even death once entering into human body.
How are endotoxins produced?
Recombinant proteins mainly rely on E.coli expression system for intracellular expression. Prior to purification, it is necessary to perform host cell lysis via high pressure homogenate or ultrasonic crushing to release protein. However, hazardous LPS are released to fermentation broth simultaneously, which constitutes the major source of endotoxin.
Apart from that, exogenous factors including production environment, operation staff, raw and auxiliary materials used will inevitably introduce endotoxins. Therefore, it is of paramount significance to restrict endotoxin content at an acceptable level to ensure the safety and effectiveness of biological products.
What are the methods to remove endotoxins?
Ion exchange (IEX) chromatography
IEX chromatography is the most significant chromatography method for the removal of endotoxin, which achieves the purpose of protein purification at the same time. Endotoxin has low isoelectric point, while most buffer solution carries large amount of negative charges. When using anion exchange (AEX) resins to perform flow-through mode purification, endotoxin will bind with positively charged ligands whereas detach from target proteins.
In the cation exchange chromatography mode, endotoxin can still be removed via flow-through due to the negative charges carried even when pH is 4.
Hydrophobic interaction chromatography
Generally speaking, endotoxin enjoys better hydrophobicity than most bio-molecules. Under high salinity condition, endotoxin will be more likely to agglutinate and can flow through when loading sample, achieving the separation of target proteins. Alternatively, when choosing low salinity, endotoxin will bind with resin while target proteins flow through. The two methods can be used to remove endotoxin.
Mixed mode chromatography
Mixed mode chromatography can simultaneously provide multiple interaction forces, including electric charge interaction and hydrophobic interaction. Endotoxin carries great amount of negative charges, which offers strong hydrophobicity. Therefore, charge exchange collision and hydrophobic interaction can be used to remove endotoxin.
Size exclusion chromatography(SEC)
Molecular weight of endotoxin monomer is about 10KD. However, aggregates of varying degrees of aggregation (10KD-1000KD) will form in water solutions with various ingredients. The formed aggregates differ significantly from major bio-molecules, therefore can be separated from target proteins via size exclusion chromatography. Nevertheless, this method can be relatively time-consuming due to the restriction of sample loading volume.
Ultrafiltration
The aggregates and endotoxin possess high molecular weight. Apart from size exclusion chromatography, effective block of endotoxin can be achieved by tangential flow membrane whose pore size is smaller than molecular weight of endotoxin, eventually remove endotoxins.
Conclusion
Endotoxin content is a key parameters in the quality control of biological products. Due to the heterogeneity of various endotoxins, no widely applicable method for endotoxin removal is available right now. Thus, a combination of removal methods can be employed in the reduction of endotoxin content.
Chromatographic resins are commonly used in purification processes to remove endotoxins from biopharmaceutical products. Therefore, it is very important to purchase high-quality chromatography resin. As one of China's top chromatography resin suppliers, we have been providing high-quality resin products to customers around the world. If you need chromatography resins, visit our product catalog.
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We are a leader in resin-based separation of China with the capability to produce chromatography resins on bio-process scale. Bestchrom offers a wide range of separation resins, pre-packed columns, lab and process scales empty columns, microcarriers, and customization services.
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