Why to test endotoxin
Because endotoxin is everywhere
Endotoxin, also known as lipopolysaccharides (LPS, molecular weight range of 3000-4000da, composed of long-chain complex polysaccharide tail and hydrophobic lipid groups (Figure 1)), is a component of the cell wall of most Gram-negative bacteria, with high thermal stability. When bacteria are growing actively or decomposing in death, lipopolysaccharide in cell wall is released into surrounding environment.
Fig. 1. Structure of lipopolysaccharide. Endotoxin, a complex lipopolysaccharide, is the active structural component of the cell wall of Gram-negative bacteria. They are composed of core oligosaccharide chain, O-specific polysaccharide side chain (O-antigen) and lipid component (lipid A).
The use of recombinant proteins, such as those produced by E.coli, or the extraction of nucleic acids from Gram-negative bacteria often encounter endotoxin pollution. When endotoxin contaminated protein / antibody / nucleic acid is transfected into cells or injected into animals, it can cause strong immune response, leading to systemic inflammatory response syndrome (SIRS) and / or sepsis. Therefore, endotoxin removal is necessary for injection drugs and biological products for human disease treatment, especially for downstream purification of gene drugs, protein drugs, antibody drugs, vaccines and other biological products.
Provisions of Chinese Pharmacopoeia
Bacterial endotoxin test (general rule 1143) includes two methods: gel method and photometric method, and the latter includes turbidimetric method and chromogenic method. Any bacterial endotoxin test can be used in any of them. Gel method and photometric method have their respective advantages and limitations:
Gel method has the advantage of simple operation and can be tested by gel method after removing interference.
The interference test of gel method is the decisive factor to determine whether the test product can be used gel method. In the interference test, the samples which have negative reaction with tachypleus amebocyte lysate should be selected for interference test. If the sample is diluted to MVD (the maximum effective dilution ratio), it still can not exclude the interference effect. Further studies should be done on the pre-treatment of the sample, and then the interference test is used to verify whether the gel method can be used.
Photometry (including turbidimetric method and chromogenic method) can quantitatively detect endotoxin content and accurately assess the relative risk of product pollution in the production process. The data of quantitative detection is not only conducive to tracking the product quality trend, but also plays a role of risk early warning, and is easier to meet the requirements of data integrity.
Because the detection limit of photometric method is wider than that of gel method, the interference sample can have a larger dilution multiplier. For some samples that can not be interfered by gel method, we can try to establish bacterial endotoxin test method by spectrophotometry.
Common experimental methods and interference analysis
Limulus amebocyte lysate is a lysate extracted from Limulus amebocyte lysate of marine animals. It is a sensitive reagent for detecting the endotoxin of Gram-negative bacteria. The experimental principle is shown in Figure 2:
Fig. 2. Cascade reaction of Limulus amebocyte lysate. LPS (endotoxin) activated plasma membrane binding factor C. Activated factor C activated factor B, activated factor B activated prothrombin and produced thrombin. After adding the chromogenic substrate AC ile Glu ala Arg PNA, the activated protease thrombin catalyzes the release of p-nitroaniline (PNA) and produces yellow. Quantitative analysis was carried out by measuring the absorbance value at 405nm. According to the standard curve, the result can be calculated. Red indicates no active enzyme and green indicates active enzyme.
Generally, the experimental process is as follows:
Thermo provides a photometric endotoxin quantitative reagent (a39552). So what is the quality of the reagent? We will verify the performance of Pierce endotoxin quantitative kit from the perspectives of sensitivity, data repeatability and anti-interference
Data sensitivity and repeatability provide two linear dynamic ranges: 0.01-0.1 EU / ml and 0.1-1.0 EU / ml (Figure 3). The difference coefficient (CV) between experiment and operator operator was 3%. High sensitivity can effectively avoid the potential false negative or false positive in endotoxin quantification.
Figure 3. Standard curve of Pierce endotoxin quantitative kit. The standard curve shows excellent linearity, R2 = 0.99. The lower standard curve range of the assay kit was 0.01-0.1 EU / ml. The standard curve of 0.1 – 1.0 EU / ml indicates that endotoxin standard and amebic cell lysate were used for culture for 14 minutes, followed by chromogenic substrate for 6 minutes. For lower concentrations, endotoxin standard and amebic lysate were used for 30 minutes, followed by chromogenic substrate for 6 minutes. The low concentration standard (0.01-0.1 EU / ml) showed consistency and reproducibility (n = 17; CV = 3%).
The compatibility of tachypleus amebocyte lysate may be affected by many factors, resulting in false negative or false positive. Include:
i. Reaction temperature, sample pH, ionic strength and metal ions (such as magnesium and calcium)
II. Serum proteins, nucleic acids, fatty acids, surfactants and chelators (such as EDTA and heparin) can cause changes in the molecular structure of endotoxin
III. nonspecific interference of (1-3) - β - D-glucan: tachypleus amebocyte lysate will react with it, resulting in false positive results
Table 1. The highest concentration compatible with the effective endotoxin test results was obtained by using the pierce colorimetric method. The listed concentrations refer to the actual concentrations in which 0.5 EU endotoxin is added to the sample without reducing the quantitative value. The dilution concentration is expressed in proportion, where 1:100 is 100 times dilution
It should be noted that the pierce assay kit (art. No. a39552) is compatible with β - glucan and does not show enhancement in samples containing 10NG / ml (1,3) - β - D-glucan.
Endotoxin removal
Ultrafiltration, polymyxin B affinity resin and chromatography based on resin or membrane are the traditional methods for endotoxin removal. These methods have some limitations in protein recovery, endotoxin binding capacity and other aspects, some of them are toxic.
Thermofisher scientific provides a high load endotoxin removal resin (88270), which is based on ε - polylysine affinity resin (a nontoxic polymer of natural lysine), showing excellent endotoxin binding capacity and protein recovery:
? the binding force can reach 2000000 EU / ml
? when the initial endotoxin level is 10000 EU / ml, the removal efficiency can reach more than 99%
Figure 4. High throughput endotoxin removal efficiency of Pierce resin. (A) Work flow of endotoxin removal and quantification. (B) Determination and removal of endotoxin in protein samples for downstream application.
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