Why is it important to check for bacterial endotoxins in drug development?

 

Impurities that need to be controlled may be generated during the production and standard storage of drugs according to established processes, and these impurities may impact the activity and safety of the drug itself. They may also cause adverse reactions in the clinical use of the drug. Therefore, impurity analysis is crucial in pharmaceutical research to drug development.

Bacterial endotoxin is a process-related impurity, one of the headache impurities in the purification of proteins, nucleic acids, polysaccharides, and other biological macromolecules, which are widely present in reagent consumables and samples. It is a significant source of contamination in biomedical tests and pharmaceutical products, and the inspection of bacterial endotoxin has become one of the critical testing items for drug quality control.

Bacterial endotoxin is a complex formed by lipopolysaccharide of the cell wall of Gram-negative bacteria, and a variety of trace proteins, which are released with biological activity when the bacteria die or disintegrate, and bacterial endotoxin is related to pyrogen. The impurities in the drug that can cause an increase in body temperature are pyrogens, mainly derived from the bacterial endotoxin of gram-negative bacteria, and the human body is susceptible to endotoxin, which can cause fever when mixed with picogram levels in each milliliter of blood.

Endotoxin is the leading cause of failure in the pyrogen testing of pharmaceuticals. Under GMP conditions, the accepted view of quality control in pharmaceutical production is that no endotoxin means no pyrogen, and control of endotoxin means control of pyrogen.

To ensure the success and reliability of biomedical tests and the safety and efficacy of biomedical products, the complete removal of bacterial endotoxins from samples and solutions is of great concern to pharmaceutical developers.

Since bacterial endotoxins can cause rapid agglutination of the blood cell lysates of a marine organism, horseshoe crab, a variety of compassionate bacterial endotoxin detection methods based on extracts of horseshoe crab blood, LAL/TAL reagents, have been derived from this. The 2010 edition of the Chinese Pharmacopoeia stipulates that the bacterial endotoxin check method is a method that uses horseshoe crab reagents to detect endotoxin or quantify bacterial endotoxin produced by gram-negative bacteria to determine whether the limit of bacterial endotoxin in the test article complies with the regulations.

Among the methods for endotoxin detection using LAL/TAL reagents, the gel method, the turbidity method, and the colorimetric method is recognized and widely used by significant biological industries. 

Colorimetric and turbidimetric methods are used for impurity analysis, and the impurity-specific colorimetric (colorimetric) and precipitation reactions (turbidimetric) can be used against standards.

However, preparing LAL/TAL reagents requires capturing rare animals, horseshoe crabs, and other methods that require using LAL/TAL reagents for endotoxin detection, such as enzyme-linked immunosorbent assay and flow cytometry are coming on the market one after another. So what are the main methods used to detect bacterial endotoxin?

1, Gel method

The gel method is to use horseshoe crab reagent to quantify the detection of bacterial endotoxin produced by gram-negative bacteria, the sample to be tested is mixed with horseshoe crab reagent, and after a while, the principle of agglutination reaction between horseshoe crab reagent and endotoxin is used to determine whether the limit of bacterial endotoxin in the test article following the regulations of a method. 

The method is easy and inexpensive to operate, but it is also less automated, more time-consuming, and prone to false positives. Gel chromatography and sol-gel methods are all impurity analysis methods.

For example, one researcher used the gel method to establish the bacterial endotoxin examination method for benzene sulfonylurea cisatracurium injection [1]. 

Methods Referring to the 2015 edition of the Chinese Pharmacopoeia (Part IV) for bacterial endotoxin examination, the inhibition of bacterial endotoxin examination by cisatracurium besylate the injection was verified by testing and dilution of the test article with diluent I instead of water for bacterial endotoxin examination (WBET) could eliminate the inhibition of endotoxin examination by Cisatracurium besilate.

The results showed that supplementing the appropriate amount of divalent ions could effectively eliminate the inhibition of bacterial endotoxin check by phenyl sulfonium cisatracurium injection. It is concluded that the maximum non-interfering concentration of 0.02 g/L can control bacterial endotoxin in benzene sulfonylurea cisatracurium injection by gel method. Medicilon provides pharmaceutical impurity analysis services, which can use techniques such as HPLC to separate impurities in regulated starting materials, intermediates, APIs, and drug products.

2, Turbidity method

The turbidimetric method to produce turbidity change by bacterial endotoxin binding to the C-factor of horseshoe crab reagent and activating coagulase to form coagulation protein. As an impurity analysis process, the turbidimetric analysis technique can also be used to help study the source of impurities in crystals and the effect of pollutants on infrared absorption and color-centered lasers.

The turbidity method is divided into the endpoint and dynamic processes according to the different measurement methods. The turbidimetric method for detecting bacterial endotoxin is simple to operate, not affected by the test environment, and has the characteristics of high sensitivity and wide detection range. However, the turbidimetric method needs to be equipped with special instruments.

For example, one researcher used the turbidimetric plan to explore the bacterial endotoxin examination of aqueous solutions of moistened burn cream after hexane extraction [2]. The method uses an additive quantitative endotoxin inoculation method to extract the burn cream endotoxin from the oil phase into the aqueous phase, and the dynamic turbidimetric method was used to determine the endotoxin recovery and to examine the effect of the extraction process on endotoxin; the gel method was also used to validate the non-interference concentration further. 

Results The recovery of endotoxin after the 6-fold dilution of the extract was 52.3%. There was no interference with the reaction of horseshoe crab reagents, and the effects of both methods for detecting endotoxin in the section were within the specified limits. Conclusion After the wet burn cream was dissolved with hexane, it was shaken and mixed with water for bacterial endotoxin examination. It was feasible to take the aqueous phase extract for bacterial endotoxin examination.

3, Colorimetric method

The colorimetric method is based on the hydrolysis of horseshoe crab tripeptide by coagulase activated by bacterial endotoxin, releasing yellow p-nitroaniline, which is detected by detecting the absorbance, or by detecting the red-azo-blue complex formed by the reaction between the released p-nitroaniline and azo reagent.

Although the colorimetric method is more complex, it is more sensitive and accurate and is suitable for endotoxin detection in recombinant protein drugs.

4, Enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay quantifies endotoxin by detecting endogenous pyrogens, such as TNF and IL-1, produced by macrophages stimulated by bacterial endotoxin. This method is simple and reproducible, but the operation is tedious, and the sugar component will stimulate the cells to produce endotoxin to interfere with the detection. The interference of the sugar component must be excluded, and the detection accuracy could be better than the horseshoe crab reagent method.

5, Flow cytometry

Flow cytometry uses monoclonal antibodies against endotoxin surface antigens, fluorescent labeling of endotoxin, and flow cytometry to detect. The method does not need to rely on endotoxin activity and can be directly detected endotoxin content.

Conducting drug impurity analysis, standardized detection, and reasonable control of impurities in drugs is related to the safety and effectiveness of drugs. As one of the critical testing items of drug quality control, the bacterial endotoxin detection method plays a vital role in ensuring the safety of clinical drug use. 

There are many methods for the detection of endotoxin. Scientific validation should be performed when determining the detection method, and the verification of the detection method should be gradually improved.

 

[1] Study of bacterial endotoxin detection method for Cisatracurium besilate injection [J].

[2] A methodological study of bacterial endotoxin examination of burn cream [J].