Chromatography Assay
The selection of a suitable endotoxin removal system is based on the properties of the bioproducts. Chromatography is likely the most widely and reliable applied method due to its ability to exploit the size, charge, hydrophobicity, nucleotide accessibility, and affinity of a biomolecule. Creative Biolabs provides chromatography assays to remove the endotoxin in specific test samples. Here we specifically focus on chromatography-based techniques including ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and gel filtration chromatography.
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Anion exchange chromatography
Anion exchange chromatography (AEC) is one of the most currently used chromatographic techniques for endotoxin removal. The negatively charged proteins compete with endotoxins for binding sites which eventually exhaust the ligand binding capacity. The presence of only net positively charged proteins will cause protein molecules to experience repulsion from the ligands and compete with ligands to capture endotoxin. Endotoxins captured by proteins will be dragged out of the column and diminish the endotoxin removal efficiency of the ligand.
Application: This method is suitable for the purification of positively charged protein.
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Affinity chromatography
Affinity chromatography is composed of triple helix (THAC), protein-DNA, immobilized metal (IMAC), boronate, polymyxin B, histamine, arginine, and histidine affinities. This method uses synthetic ligands for specific elution and involves the impregnation of poly(ε-lysine) into cellulose beads which provide greater endotoxin selectivity. Pore size plays a major role in particle selectivity. The current drawbacks of affinity chromatography are low yield and high salt concentration requirement for substance elution.
Application: This method is used to separate endotoxins from target molecules using highly specific interactions between endotoxins and a ligand bound to a stationary phase.
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Hydrophobic chromatography
The immobilized hydrophobic ligands of hydrophobic chromatography interact with nonpolar protein surfaces through van der Waals forces for high endotoxin removal. Endotoxin and protein are adsorbed onto the ligands and later separated using salt addition based on gradient elution. Ammonium sulfate and sodium citrate are commonly applied as binding buffers.
Application: This method explores differences in hydrophobicity for plasmid purification and captures pDNA under high-salt conditions due to the predominant hydrophilic feature of pDNA; Immobilized hydrophobic ligands interact with nonpolar protein surfaces through van der Waals forces for high endotoxin removal; also functions as an analytical tool for pDNA quality control and monitoring.
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Gel filtration chromatography
Gel filtration chromatography, also well known as size exclusion chromatography (SEC), is the gold standard for separating protein aggregates from their monomeric form. SEC uses composite polyacrylamide as the highly porous column. Compared to the other chromatographic methods, SEC has limited pDNA capacity and selectivity. Both SEC and ultrafiltration require product and contaminant to have a large size difference for effective endotoxin removal. Ultrafiltration is used if a protein is not present, which is capable of removing large endotoxin aggregate with alkanediol as one of the many agents used for effective endotoxin removal.
Application: For a simple, inexpensive, and reproducible pDNA or protein purification, SEC can be considered.
Features of Different Methods
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Method
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Features
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Anion exchange chromatography
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It has rapid separation, wide selection of AEC media, sodium hydroxide (NaOH) sanitization, and does not require any solvents.
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Affinity chromatography
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Affinity adsorbents include immobilized L-histidine, poly-L-lysine, poly (γ-methyl L-glutamate), and polymyxin B.
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It is highly specific and efficient for endotoxin removal combined with excellent target recovery.
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Hydrophobic chromatography
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This method interacts with non-polar enzyme surfaces through van der Waals forces for endotoxin removal.
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Gel filtration chromatography
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It uses composite polyacrylamide as the highly porous column.
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It can remove endotoxins from product solutions if the products are of low molecular weight.
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It is limited to those proteins whose solubility is not largely affected by the presence of high concentrations of calcium ions.
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Creative Biolabs will use one or a combination of the above methods to ensure the maximum efficiency of your endotoxin removal project. We offer a corresponding endotoxin removal service and you can purchase the corresponding endotoxin removal kit and related accessory products according to your needs. We guarantee that all instruments, water, reagents, and consumables used in the experiment are free of endotoxins. If you are interested in our endotoxin removal assay, please feel free to contact us or directly send us an inquiry.
References
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Mason, S.; et al. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Authorea. 2019.
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Clarence, M.; et al. "Chromatographic removal of endotoxins: A bioprocess engineer's perspective", International Scholarly Research Notices. 2012.
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Acıkara, Ö.; et al. Affinity chromatography and importance in drug discovery. 2013.
For Research Use Only | Not For Clinical Use
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