In the research, development, and production processes of biologics, endotoxin limit detection is a crucial link in ensuring product safety and effectiveness. Endotoxins, as components of the cell walls of Gram-negative bacteria, can cause severe reactions such as fever, shock, and even life-threatening situations if they enter the human body, even in trace amounts. Therefore, accurately detecting the endotoxin content in biologics and strictly controlling it within the specified limits is a necessary measure to safeguard the safety of patients' medication. The following explores the practical key points of endotoxin limit detection through detection examples of different types of biologics.
I. Endotoxin Detection of Vaccine Biologics
(I) Inactivated Vaccines
Take the inactivated influenza vaccine as an example. Its production process involves a series of steps such as virus cultivation, inactivation, and purification. After purification is completed, endotoxin limit detection of the vaccine is required in accordance with relevant US regulations. Due to the complex composition of the vaccine, which includes various substances such as virus lysates, buffers, and adjuvants, these components are highly likely to interfere with the detection results. Therefore, samples must be pretreated before detection. The dilution method is usually adopted, using bacterial endotoxin test water to dilute the vaccine samples to an appropriate multiple to reduce the concentration of interfering substances.
The US Food and Drug Administration (FDA) has strict regulations on the endotoxin limit of inactivated influenza vaccines, generally requiring it not to exceed 6 ng/mL. In the actual detection process, the TAL/LAL Reagent gel method is first used for preliminary screening. The TAL/LAL Reagent gel method is easy to operate and can quickly determine whether endotoxins are present in the sample. If the gel method test result is positive, the kinetic turbidimetric method is then further used for quantitative detection. The kinetic turbidimetric method uses instruments to accurately measure the rate of turbidity changes during the reaction between endotoxins and TAL/LAL Reagent, thereby achieving precise determination of the endotoxin content and effectively ensuring the accuracy of the detection results.
(II) Genetically Engineered Vaccines
For example, in the production of recombinant COVID-19 vaccines, genetic engineering technology is used to express viral antigen proteins. Such vaccines are characterized by sophisticated production processes and extremely high requirements for impurity control. When performing endotoxin limit detection, considering that substances such as host cell proteins and residual nucleic acids that may be present in the vaccine may interfere with the detection, the samples will first be further purified by methods such as ultrafiltration and chromatographic separation to remove interfering substances. In terms of detection methods, the recombinant factor C method, which has high sensitivity and good accuracy, is preferentially selected. This method uses the recombinant factor C of the horseshoe crab coagulation cascade reaction. After specifically binding to endotoxins, it triggers a fluorescent reaction, and the endotoxin content is quantified by detecting the fluorescence intensity. According to relevant standards, the endotoxin limit of recombinant COVID-19 vaccines needs to be strictly controlled below 0.2 EU/mL to ensure vaccine safety.
II. Endotoxin Detection of Blood Products
(I) Plasma Protein Products
Human albumin is a common plasma protein product. Since its raw material comes from human plasma, there is a potential risk of endotoxin contamination. During the production process, after multiple processes such as low-temperature ethanol separation, ultrafiltration, and virus inactivation, strict endotoxin limit detection must be carried out. Due to the certain viscosity of the human albumin solution, it may affect the reaction between endotoxins and detection reagents. Therefore, special dilution methods will be used during detection, and the reaction time will be appropriately extended. The kinetic chromogenic method is usually used as the detection method. This method activates the enzymes in the TAL/LAL Reagent through endotoxins, causing the chromogenic substrate to react, and quantifies the endotoxin content according to the rate of color change. According to industry standards, the endotoxin limit of human albumin should not exceed 0.01 EU/mL to ensure clinical safety.
(II) Coagulation Factor Products
Coagulation factor VIII is a key drug for the treatment of hemophilia. Its production process is complex, with extremely high requirements for purity and safety. When performing endotoxin limit detection, since the activity of coagulation factor products is easily affected by detection conditions, conditions such as temperature and pH value need to be strictly controlled during the detection process. To avoid interference from other components in the sample, methods such as affinity chromatography and ion-exchange chromatography are used for sample pretreatment. High-sensitivity TAL/LAL Reagent with a sensitivity of up to 0.005 EU/mL is used for detection. A combination of the gel method and the photometric method is adopted. First, the gel method is used for rapid screening. If the result is suspicious, the photometric method is used for precise quantification to ensure that the endotoxin content of coagulation factor VIII products meets the standard of not exceeding 0.03 EU/mL.
III. Endotoxin Detection of Cell Therapy Products
(I) CAR-T Cell Therapy Products
CAR-T cell therapy products are emerging biotherapeutic means in recent years. Their preparation process involves multiple links such as collection, genetic modification, and expansion culture of patients' T cells. Since various substances such as culture media and serum are required during the in vitro cell culture process, these components may introduce endotoxin contamination. When performing endotoxin limit detection, the challenge is that the cells themselves may affect the detection results. Therefore, the cells need to be separated from the detection samples first. The method of low-speed centrifugation is used to precipitate the cells, and the supernatant is taken for detection. The recombinant factor C method is selected as the detection method. This method can quickly and accurately detect low concentrations of endotoxins. According to relevant research and clinical requirements, the endotoxin limit of CAR-T cell therapy products should be controlled within 0.1 EU/mL to ensure that patients will not experience serious adverse reactions caused by endotoxins during treatment.
(II) Mesenchymal Stem Cell Therapy Products
Mesenchymal stem cells have broad application prospects in tissue repair, immune regulation, and other aspects. During their preparation and expansion processes, there is also a risk of endotoxin contamination. Since the culture system of mesenchymal stem cells contains various components such as cytokines and growth factors, these substances may interfere with endotoxin detection. Before detection, methods such as filtration and centrifugation are used to remove cells and most interfering substances. During detection, a combination of the gel method and the kinetic turbidimetric method is used. First, the gel method is used for preliminary judgment. If the result is positive, the kinetic turbidimetric method is then used for quantitative analysis. Currently, there is no unified standard for the endotoxin limit requirements of mesenchymal stem cell therapy products. However, in actual production, the endotoxin limit is usually controlled at around 0.2 EU/mL to ensure product safety and effectiveness.
There is a wide variety of biologics. Due to the differences in production processes and compositional characteristics of different types of biologics, they face different challenges in endotoxin limit detection. As can be seen from the detection examples of the various biologics above, in the actual detection process, appropriate detection methods and pretreatment means need to be selected according to product characteristics, and relevant standards and specifications need to be strictly followed to accurately detect the endotoxin content, ensure the quality and safety of biologics, and safeguard the health of patients.
