Endotoxin removal is not only a crucial step in protein expression and plasmid extraction, but also a very difficult problem. This paper summarizes the methods of endotoxin removal and the matters needing attention in each method.
Endotoxin is a lipopolysaccharide in the cell wall of gram-negative bacteria.
Endotoxin is released only when the bacteria die and dissolve or destroy the bacterial cells artificially, so it is called endotoxin.
All substances that can cause pyrogen reaction are called pyrogens.
Bacterial endotoxin is the main pyrogen in drugs. Generally speaking, bacterial endotoxin is pyrogen, but pyrogen is not equal to bacterial endotoxin.
| Physicochemical Properties of Bacterial Endotoxin |
| ·Heat resistance: 250 high temperature dry baking for one Hour for complete inactivation. |
| ·Water solubility: It is soluble in water. In production, it can be washed with non-thermal raw water to remove pyrogen. |
| ·Non-volatility. |
| ·Adsorbability: Adsorbable by activated carbon. |
| ·Damaged by acid and alkali: Immersed in 0.1M HCl or 0.1M NaOH for 4 hours. |
| ·Destruction by oxidant: 30% hydrogen peroxide soaked for 4 hours, completely destroyed. |
| Removal of endotoxin from utensils |
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| a.dry method ·Applicable object: Containers used in the production of heat-resistant articles such as glass products, metal products, etc. ·Treatment methods: 180 3 - 4 h or 250 30 min - 2 h ·Attention: 1.Before drying, it is better not to have water droplets on heated glassware, otherwise it may damage the glassware. 2.After baking, glassware should not be taken out of the electric oven immediately. It should be taken out after the temperature is moderately lowered, so as not to damage the glassware because of the large temperature difference. 3.The utensils should not contain paper or plastic products in order to avoid burning or melting during baking. |
b.Chemical degradation method ·Applicable object: Mainly used for removing endotoxin from glass, plastic and other macromolecule material utensils. Treatment methods: commonly used 3-5% hydrogen peroxide, potassium dichromate sulfuric acid cleaning solution (potassium dichromate: sulfuric acid: water common ratio 1:1:10 or 1:2:8), 0.1M HCl or 0.1M NaOH soaking removal. Generally treated for more than 4 hours. ·Attention: Wear protective equipment to prevent direct skin contact. |
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| Removal of Endotoxin in Solution | |||
| method | principle | advantage | shortcoming |
| liquid phase separation | Some detergents, such as Triton X-114, sodium deoxycholate can bind to the lipid fraction of endotoxin and extract endotoxin by liquid phase separation so that the latter can be effectively removed. | The removal rate of endotoxin is high, and the activity of active ingredients is not affected. This method is simple, efficient, cheap and suitable for large-scale application. It is commonly used in our daily protein purification. | After endotoxin removal, there will be trace detergent residues in the sample. |
| molecular sieve method | Molecular sieve is a method based on the network structure of gelatin and separated by molecular size. Because the molecular weight of protein and endotoxin is quite different, the use of molecular sieves can effectively remove endotoxin. | Removal effect is obvious. | Each processing volume is small and the processing time is long. |
| ion Exchange Chromatography | When the solution pH > 2, the endotoxin has a negative charge. Therefore, endotoxin has strong binding with anion exchange medium Q or DEAE Sepharose Fast Flow. The endotoxin on the column can be removed by high salt buffer or NaOH after elution of the target protein. | low cost and large adsorption capacity | But it is not suitable for the presence of other negatively charged substances in solution. |
| affinity chromatography | An affinity medium was synthesized by immobilizing appropriate ligands on chromatographic substrates to specifically bind endotoxin. GenScript binds polymyxin B (PMB) to Sepharose FF to adsorb endotoxin specifically. | high efficiency and selectivity | relative cost is higher |
| ultrafiltration | Ultrafiltration membranes can be used to remove endotoxins from solution because endotoxins have relatively high molecular weight. | Simple operation and large processing capacity. | The pressure during operation is high. It is not suitable for samples containing relatively large molecular weight components. Because the pyrogen can be removed while retaining or adsorbing the active ingredients in the sample, the yield of the product will be greatly affected. |
| adsorption method | Activated carbon is used to remove endotoxin because of its relatively high molecular weight. It is suitable for removing endotoxin in solution or water with relatively simple components. The usual dosage of activated carbon is 0.1%-0.5%. | The cost is low and the processing capacity is large. | Activated carbon has poor selectivity and is easy to adsorb active ingredients. The residual activated carbon in purified solution is not easy to remove, and the heavy metals it may contain may cause sample pollution, so it is seldom used at present. |
| distillation | This method can be used to produce deheat source water for injection or washing water. | Removal effect is obvious. | high cost |
| hydrophobic chromatography | Lipid A part of endotoxin has strong hydrophobicity, but it will agglomerate under high salt and can not be attached to hydrophobic chromatography column. Therefore, the hydrophobic medium that can bind the target protein can be chosen to remove endotoxin. | Suitable for samples with high salt concentration. | At present, the technology is not mature enough, and further exploration is needed. |
| As a commonly used method for endotoxin removal, attention should be paid to liquid phase separation. | ||
1.Application of Liquid Phase Separation to the Removal of Endotoxin in Protein Purification. |
2.Application of liquid phase separation to the removal of endotoxin from a large number of samples. | |
| a.After the protein was sampled, the column was slowly eluted with pre-cooled basic solution + 1% Triton X-114 until the value of A280 remained unchanged. b.The column was washed with pre-cooled de-endotoxin basic solution until the A280 value reached the baseline and remained stable. c.The eluents were eluted with gradient endotoxin-free eluents and collected in endotoxin-free collecting tubes. d.This method is widely used, such as purification by Ni column, GST column and ion exchange column. e.The purification process should be carried out in a clean environment as far as possible, and the purification time should not be too long. f.The pipette, gunhead and other equipment used should be endotoxin-free. |
a.Triton X-114 with final concentration of 1% was added to a large number of protein samples. It was well blended and bathed in ice for 5 minutes to form one phase. b.Incubation temperature exceeded its cloud point at 21 ~37 ~C and stratification was observed. c.After obvious stratification appeared in the solution, the supernatant was absorbed at room temperature for 12 000 r/min and centrifuged for 5 min. d.In order to achieve better removal effect, it can be repeated twice. e.The concentration of Triton X-114 can be increased to 2% if the endotoxin content of the sample is high. f.If the stratification is not obvious or slow after heating up to 37 C, it can be incubated at 56 C. g.This method is not suitable for removing endotoxin from membrane proteins. |
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| Configuration of Deendotoxin Reagent |
a.Before dissolution, the solid reagent was baked for 4-5 hours at 180 C to remove water and endotoxin from the reagent. This method is suitable for reagents with melting point higher than 180 C. If the melting point of the reagent is below 180 C, it is often baked overnight at 80 C. b.Dissolve the baked reagent in water to remove endotoxin, fix volume and adjust pH. c.The endotoxin content of the reagent is usually 0.01EU/ml. |
| Common reagents with melting point below 180 C or decomposition at high temperature: 1. Tris (168-172 C), 2. Urea (132.7 C), 3. Imidazole (89-91 C), 4. Glycerol (17.8 C), 5. EDTA (150 C dehydroxylation) 6. SDS (165 C) 7. Sodium sarcosinate (120 C) |
Common reagents that need to be baked before adding：(Dangerous goods, volatile odors) 1. DTT (42-43 C) 2. β-mercaptoethanol (<-100 ℃) |
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