The heat source refers to the substances that cause the animals to develop fever after being injected into their bodies. These mainly include bacterial fragments, large molecules of Gram-negative bacterial lipopolysaccharide (LPS), and small molecules of lipoteichoic acid. Lipopolysaccharide is the main component of the cell wall of Gram-negative bacteria, with its relative molecular mass ranging from several thousand to several hundred thousand; the molecular weight of lipoteichoic acid is approximately 2000 daltons.
Endotoxin is the general term for lipopolysaccharide and lipopolysaccharide A. Endotoxin is not a protein. Even when heated at 100℃ for 1 hour, it will not be destroyed. The moist heat at 115℃ for 30 minutes can only destroy about 25% of the pyrogenic substances.
The common methods for removing endotoxins from pipes and containers are dry heat treatment and alkaline washing. At 180℃ for 3 to 4 hours or at 250℃ for 1 to 2 hours, they can be dried and baked, or they can be boiled with strong alkali, strong acid or strong oxidizing agent for 30 minutes.
Endotoxins can cause fever and even shock in the human body. Therefore, injectable products pay special attention to the removal and limit of endotoxins. Different materials have different limit requirements. Refer to the Chinese Pharmacopoeia (ChP 2020), purified water for injection: 0.25 EU/ml; recombinant human growth hormone for injection: 5 EU/mg; recombinant insulin: 10 EU/mg; ceftriaxone sodium: 0.2 EU/mg; metronidazole injection: 0.35 EU/ml; large-volume infusion: 0.25 EU/ml.
For removing endotoxins from materials, an appropriate removal process should be selected based on the physical and chemical properties of endotoxins.
Endotoxins carry a negative charge. Due to the hydrophobic nature of the lipid A component in the lipopolysaccharide structure, endotoxins tend to exist in a high-molecular polymer form and are difficult to dissolve in water. Therefore, the removal of endotoxins from the material is commonly achieved through methods such as activated carbon adsorption, anion exchange resin method, hydrophobic chromatography, ultrafiltration, or nanofiltration.
The control of endotoxins in materials generally involves two aspects. Firstly, it is necessary to ensure that no microbial contamination is introduced during the production process. Secondly, an appropriate endotoxin removal process should be selected based on the properties of the materials.
Small water-soluble molecules can remove endotoxins and reduce the heat source content. As a new process and technology, the ultrafiltration membrane method is the most commonly used method in the pharmaceutical industry. Engineering experience shows that by selecting the appropriate ultrafiltration membrane accuracy, material concentration, and filtration temperature, the efficiency of endotoxin removal can be guaranteed.
For macromolecular products, column chromatography (anion exchange resin, hydrophobic chromatography filler, specific affinity filler) is commonly used. For some special products, organic solvent method and surfactant method are adopted.
The higher limit requirements for endotoxins often necessitate the use of multiple methods in combination, such as pretreatment + ultrafiltration, ultrafiltration + adsorption, or two-stage ultrafiltration filtration, etc.
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