Endotoxin Removal and Detection

Endotoxins, also known as lipoglycans and lipopolysaccharides (LPS), are a part of the outer membrane of the cell wall of Gram-negative bacteria. It is extremely difficult to remove them by simple sterilization, and as a consequence, they form the major contaminants in commercially available proteins or biologically active substances. Endotoxins can initiate a strong immune response such as endotoxin shock, organ failure, tissue injury, disseminated intravascular coagulation, and even death. Due to the harmful effects of endotoxin, the FDA has limited the concentration of endotoxin in medical devices to <0.5 EU/mL.

Toxicity of Endotoxin

The physiological activities of endotoxin are mediated primarily by the Lipid A which can stimulate the mammalian immune system. After interacting with specific receptors on the immune cells, high concentrations of cytokines and other molecules of immunological significance are released that result in a wide spectrum of nonspecific pathophysiological reactions such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock, and even death.

Furthermore, endotoxins can strongly influence transfection of DNA into primary cells and sensitive cultured cells, and increased endotoxin levels lead to sharply reduced transfection efficiencies. Using endotoxin-free plasmid DNA for gene therapy applications is extremely important since the high toxicity of endotoxins cause strong biological effects in cell cultures or animals.

Endotoxin Removal and Detection Services

With years of research experience in the field of recombinant protein expression, Biologics International Corp (BIC) deeply understands that removal of endotoxins from your biological sample is extremely important for downstream applications. We will be your reliable partner in effectively reducing the endotoxin level below 0.01 EU/µg and meeting strict requirements for downstream animal and cell experiments. Contact us for project quotations and more detailed information.

Our endotoxin removal service includes gene synthesis, protein expression, protein purification, and endotoxin removal and detection. All you need to do is simply provide the protein sequences. Endotoxin control and removal starts from protein preparation experiment to ensure low levels of endotoxins. All the vessels, the instruments, water, reagents, and liquid move straws used in the experiments require endotoxin removal.

Gene synthesis

Protein expression

Protein purification

Endotoxin removal

Endotoxin detection

Methods of Endotoxin Removal

Endotoxin removal is one of the most difficult tasks in downstream processes during protein purification. At BIC, our experienced scientists will develop a custom protocol for your target protein after estimating the endotoxin removal efficiency and the sample recovery rates. We will use a combination of several of the following methods to assure the highest efficiency.

• Endotoxin affinity chromatography

The high selectivity of affinity chromatography eliminates the need for multiple purification steps and reduces production costs. Meanwhile, this method can remove endotoxins with highly specificity and efficiency achieving excellent target recovery.

• Ion exchange chromatography

This method has the advantages of rapid separation, wide selection of AEC media, and sodium hydroxide (NaOH) sanitization and does not require any solvents.

• Hydrophobic chromatography

The hydrophobic column interacts with nonpolar protein surfaces through van der Waals forces for endotoxin removal.

• Gel filtration chromatography and ultrafiltration

It uses composite polyacrylamide as the column, which is highly porous. Ultrafiltration results in relatively good endotoxin clearance from product solutions when the products are of low molecular weight.

• Triton X-114 phase separation

This method involves the separation of aqueous surfactant solution into micelle-rich and micelle-poor regions through excluded-volume interactions. External agent notably Triton X-114 is needed to maintain the inherent biological activity of the protein while reducing endotoxin level by 100-fold.

Methods of Endotoxin Detection

The limulus amoebocyte lysate (LAL) assay is the FDA-approved method for endotoxin detectionis, which is also the most commonly used approach. There are three forms of the LAL assay, each with different sensitivities.

• LAL gel-clot assay

This method can detect endotoxin concentrations down to 0.03 EU/mL. However, it can only be applied to semiquantitative detection.

• LAL turbidimetric assay

Turbidimetric assay can be used for quantitative detection with an accurate result. Its detection range is from 0.01 to 100.0 EU/mL.

• LAL chromogenic assay

Like LAL turbidimetric assay, chromogenic assay is widely used for quantitative and accurate endotoxin detections. This method can detect down endotoxin concentrations to 0.01 EU/mL.

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