Removing endotoxin is particularly important for biological products. Experiments have shown that CytoNiche's 3D TableTrix microcarriers can meet relevant safety requirements and quality standards at all levels.

Endotoxin is a component in the cell wall of Gram-negative bacteria, called lipopolysaccharide (LPS). LPS consists of lipid A, core polysaccharide, and O antigen. Among them, lipid A is a glycophospholipid, which is the toxic part of endotoxin that related to its pathogenicity. The lipid A structure of different Gram-negative bacteria is basically similar, and the endotoxin-related toxic effects are roughly similar in the infections caused by lipid A. The O antigen is the highly variable region that can trigger the immune response. The core polysaccharide mainly plays a linking role, and sometimes also shows a specific antigenicity.

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【The Importance of Endotoxin Testing】

Endotoxins can be released in large quantities through lysis of bacteria after death, and can also be released in the form of cell wall belbing of viable bacteria. Ubiquitous in the environment, endotoxin features small size, high chemical stability, and high thermal stability. It is not easy to be eliminated or inactivated through filtration, heating and chemical methods, but can be removed by ultrafiltration, dry heat and chemical degradation methods.
Endotoxin intake is not easily detected in cell culture, and the source of contamination usually comes from microcarriers for cells, serum, culture medium, washing solution, etc. The toxic effect of endotoxin is weak, but the human body is extremely sensitive to endotoxin.

Its influence mainly includes:

1. Changing the shape of cells, such as causing changes in the shape of liver cells and reducing their ability to absorb six-carbon sugars.

2. Activating cells, producing message-transmitting substances, and affecting their downstream biological pathway responses. For example, endotoxin can activate macrophages to secrete cytokines such as tumor necrosis factor (TNF), interferon (IFN), and interleukin 1 (IL-1), which act on the thermoregulatory center in the host's hypothalamus to increase body temperature and cause fever.

3. Inhibiting enzyme activity, such as Angiotensin Converting Enzyme.

4. Promoting or inhibiting cell division. The difference in effect may be related to the concentration of endotoxin.

5. Inhibit the egg fertilization rate of In Vitro Fertilization.

6. Other aspects: such as affecting cell adhesion, reducing the binding capacity of liver cells to insulin, reducing the loss of potassium ions in cells, etc.

【Endotoxin Testing Method】

The endotoxin testing method included in the Chinese Pharmacopoeia is the LAL test (the LAL contains factor C, factor B, pro-clotting enzyme, coagulogen, etc.). There are two types of methods for LAL test, namely gel-clot test and photometric method. Any one of these methods can be used for the test article detection. When the determination results are in dispute, unless otherwise specified, the gel limit test results shall prevail.

1. Gel-clot Test

Gel-clot test is a method for limit test or semi-quantitative test of endotoxin using the principle of agglutination reaction between LAL and endotoxin, and the method is easy to operate.
Endotoxin can activate factor C to produce a series of cascade reactions after being in contact with LAL, and it can also activate factor G through (1,3)-β-D-glucan to produce cascade reactions, which will make LAL into gel. Therefore, it is necessary to exclude the interference of samples containing (1,3)-β-D-glucan when using this method to detect endotoxin.

The picture comes from the Internet

2. Photometric Method: Turbidity Method and Chromogenic Matrix Method

①The turbidinetric method includes dynamic turbidity method and end-point turbidity method: It is a method to measure the endotoxin level through the turbidity change during the reaction between the LAL and the endotoxin.

♢Dynamic turbidity method is a method to detect the reaction time required for the turbidity of the reaction mixture to reach a preset absorbance or transmittance, or to detect the rate of increase in turbidity.

♢End-point turbidity method is a method for determining endotoxin level based on the quantitative relationship between the endotoxin concentration in the reaction mixture and its turbidity (absorbance or transmittance) at the end of the incubation.

② The chromogenic matrix method includes dynamic chromogenic method and end-point chromogenic method: It is to detect the amount of chromophore produced by the coagulase generated during the reaction between LAL and endotoxin, so as to detect the endotoxin level. The test method, which calculates the endotoxin level according to the color of the product, is also known as the colorimetric method.

♢Dynamic chromogenic method is a method to detect the reaction time required for the chromaticity of the reaction mixture to reach a predetermined absorbance, or to detect the chromaticity growth rate.

♢End-point chromogenic method is a method for determining endotoxin level based on the quantitative relationship between the endotoxin concentration in the reaction mixture and the amount of chromophore released at the end of the incubation.

Whether the test articles can be detected by photometric method must be determined by interference test. The photometric method can be used to determine the trend of interference from the recovery rate, which is especially advantageous for samples of research nature (such as new products).
Since the detection range of the photometric method is wider than that of the gel-clot method, the samples with interference can be diluted with a larger dilution factor. For some samples in which the interference cannot be excluded by the gel-clot method, the photometric method can be used to establish a bacterial endotoxin testing method.

【Problems facing endotoxin testing】

Limulus reagent test is currently the most commonly used detection method, which is accurate, sensitive, simple and fast. At present, the annual demand for LAL in our country is about 10 million. In recent years, due to the ecological destruction near the shallow sea and human predation, the number of horseshoe crabs has dropped sharply. On February 5, 2021, the State Forestry and Grassland Administration and the Ministry of Agriculture and Rural Affairs issued an announcement to list the raw material "limulus" (tachypleus tridentatus and carcinoscorpius rotundicauda) used in LAL as national second-class protected animals. This means that the raw materials of endotoxin testing reagents will be strictly controlled.

The picture comes from the Internet

【New Method for Endotoxin Testing】

The "9251 Guidelines for Using the Test for Bacterial Endotoxin" in the Chinese Pharmacopoeia (2020 Edition) proposes that the artificially synthesized recombinant factor C method, micro-gel method, etc. can be used for endotoxin testing to reduce the use of LAL. Recombinant factor C is a chemically synthesized reagent with more stable properties and relatively better homogeneity. Bacterial endotoxin level is determined based on the quantitative relationship between the endotoxin concentration in the reaction mixture and its fluorescence value at the end of the incubation. The micro-gel method is roughly the same as the gel-clot method. The difference is that the amount of LAL used in the micro-gel method is only one-tenth of that in the original method, which greatly reduces the use of LAL.

【Endotoxin Testing in Cell Culture of CytoNiche 3D TableTrix® Microcarrier Tablet】

The removal of endotoxin is necessary for injectable drugs and biological products for human disease treatment, and it is especially important for the quality control of cell therapy products.

Using microcarriers for large-scale cell preparation, whether the endotoxin level in the final harvested cell product meets the safety standards for clinical applications is also an issue that needs to be considered in quality control during large-scale cell preparation.

The core product of CytoNiche, the 3D TableTrix® Microcarrier Tablet, is designed for low endotoxin. This product has completed the filing of the qualifications for pharmaceutical excipients from the Center for Drug Evaluation (CDE) of the National Medical Products Administration (F20210000003 and F20210000496) and U.S. FDA/DMF (DMF: 35481).

We used 3D TableTrix® Microcarrier Tablet combined with 125mL, 500mL and 5L series of CytoNiche bioreactors for cell suspension culture. The endotoxin levels in cell supernatant and cell suspension (cell concentration: 1×106) after dynamic culture were detected in multiple batches. The results showed that the endotoxin levels in the cell suspension and cell culture supernatant after large-scale culture on 3D TableTrix® Microcarrier Tablet were less than 0.25 EU/mL.

Picture: Positive groups in the front row, negative groups in the back row

Experimental results: In the experiment, the gel clots were formed in the positive control groups, but not in the negative groups, which meant that this experiment was successful, as shown in the figure above.

During the test article detection, the gel clot was formed in the positive test article, but not in the negative test article, which was slipped off after inversion of 180°, and the data showed that the endotoxin level of the test article detected in this experiment was less than 0.25 EU/mL. All data have proved that the large-scale cell expansion process using 3D TableTrix® Microcarrier Tablet will not bring endotoxin interference to the final cell product.

CytoNiche 3D TableTrix® Microcarrier Tablet can meet the regulatory requirements for the safety of raw materials used in large-scale cell expansion CMC from the aspects of standard establishment, raw material selection and carryover testing method, and meet the quality standards for the clinical application of cell therapy final products.