1. What is endotoxin?
Endotoxin is a type of lipopolysaccharide (LPS) that originates from the outer membrane of Gram-negative bacteria. The outer lipid component of the cell wall is entirely composed of endotoxin molecules and is released upon cell death or decomposition. LPS has three structures consisting of bacterial-specific polysaccharides, non-specific core polysaccharides, and lipid A. The monomeric molecule of endotoxin has a molecular weight of around 10 kDa, and in different components of aqueous solutions, it can form large polymer aggregates with a maximum weight exceeding 1000 kDa. Lipid A is the main functional group responsible for the various biological activity or toxic responses of endotoxin. This group is not species-specific, and therefore the lipid A structures of various bacteria are similar, resulting in similar toxic responses including fever, changes in blood flow dynamics, disseminated intravascular coagulation, and shock. O-specific polysaccharides are located in the outermost layer of the bacterial cell wall and are composed of several repeating units of oligosaccharides. The type and number of polysaccharides determine the specificity of bacterial species and type, as well as the common antigenicity between different bacteria. It also participates in the antibody dissolution of bacteria.
1.1 Properties of Endotoxin
Endotoxin is not a protein and is highly thermally stable, as it can withstand heating at 100°C for 1 hour without being destroyed. It can only be partially destroyed by wet heating at 115°C for 30 minutes, which can break down about 25% of its thermal nucleotide. Its biological activity can only be destroyed by dry roasting at 180°C for 3-4 hours or at 250°C for 1-2 hours, or by heating with strong alkalis, acids, or oxidizing agents at boiling temperature for 30 minutes.
Endotoxin carries a negative charge and tends to exist in the form of high molecular weight polymers, which are insoluble in water due to the hydrophobicity of the lipid A structure in lipopolysaccharides. It is stabilized by ions such as Ca2+ and Mg2+ attracted to the negatively charged lipopolysaccharide. Its molecular weight typically ranges from several hundred thousand to tens of millions of daltons.
1.2 Forms of Endotoxin
- Monomer
- Micelle
- Vesicle
2. Endotoxin Production
Genetically engineered proteins are usually produced on a large scale through fermentation in bacteria, yeast, mammalian cells, or insect cells as hosts, with Escherichia coli serving as the most commonly used expression system. Proteins expressed in E. coli are usually intracellular and the bacterial cells need to be disrupted by high-pressure homogenization, ultrasonic fragmentation, or low-pressure infiltration before purifying the protein to release the protein. At the same time, lipopolysaccharides inside the cell wall are also released in large amounts into the buffer, and a wet bacterial concentration of 10% can usually produce several thousand EU/mL of endotoxin, which is the main source of endotoxin.
Although yeast expression systems or CHO (Chinese hamster ovary cell line) systems do not themselves produce endotoxin, product endotoxin contamination can occur during production due to factors such as raw material and production environment, as well as personal operation, which is another source of endotoxin.
How to prevent endotoxin pollution during production?
- Prevention of endotoxin pollution in crude purity: Whether it is yeast, mammalian cells, insect cell expression system, or E. coli expression system, it is very important to effectively prevent endotoxin pollution by using various methods through various channels during the production process. The former does not produce endotoxins itself, and as long as endotoxin pollution can be effectively prevented during the production process, qualified products can be obtained; the latter produces endotoxins, but effective measures have been taken in the initial purification process, which can greatly reduce the endotoxin content.
- Prevent exogenous endotoxin contamination through aseptic operation and control of the cleanliness of the operating area: First of all, the production workshop must be a GMP-compliant clean room and various operations are carried out in different clean areas. For example, fermentation and crude purity should be carried out in a 100,000-level area, and refinement should be carried out in a 10,000-level area. Formulation and packaging must be carried out in a 100-level room or under a laminar hood. For any non-closed system operation, an aseptic operation should be adopted. If the degree of system automation is high, remote-control operation or less manual operation can be used.
- Strict and effective treatment must be carried out for any solutions, containers, production equipment, pipelines, and production systems that come into direct contact with the products to remove heat sources. Fresh injection water or sterilized injection water prepared by distillation or reverse osmosis method usually does not contain heat sources, and this is the basis for cleaning equipment and preparing solutions. Glass containers and stainless-steel products are a type of production equipment that can be baked at 180℃ for 3-4 hours or 250℃ for 1-2 hours to remove heat sources. Another type of equipment is rubber and plastic products, such as rubber plugs and hoses, which can be boiled in 0.1M hydrochloric acid for 30 minutes or soaked in 0.1M sodium hydroxide for more than 4 hours. In case of emergency, it can be boiled for 30 minutes and then rinsed with fresh injection water before high-pressure sterilization. For some key steps in biological pharmaceutical production, sterile and heat source-free disposable equipment can be used to reduce labor and prevent pollution caused by incomplete cleaning. Fresh injection water without heat sources must be used for solution preparation, and the entire process must be completed within 3 hours and high-pressure sterilized promptly. For some solutions that cannot be sterilized, heat sources can be removed by ultrafiltration. For example, when PBS is sterilized under high pressure, it will produce pyrophosphate with ultraviolet absorption. This will interfere with the chromatographic profile when using phosphate buffer (PBS) for column chromatography. Such problems have occurred in the production and testing of interferon. Ultrafiltration with ultrafiltration membranes with a cutoff molecular weight (NMWL) of 10 kDa can effectively remove heat sources from the solution.
3. Detection of bacterial endotoxins
The bacterial endotoxin detection method uses Limulus reagents to detect or quantify the bacterial endotoxins produced by Gram-negative bacteria, which is a method to determine whether the limit of bacterial endotoxins in the test sample meets the specified requirements. The reaction mechanism of the Limulus reagent method is that bacterial endotoxins activate a series of coagulation enzymes in Limulus hemolymph under the participation of divalent cations. There are two methods included, namely the gel method and the photometric method, which includes turbidimetry and chromogenic substrate methods. Any of these methods can be used for detection. When there is a dispute in the results, unless otherwise specified, the gel method result shall prevail. The Limulus reagent method has the advantages of being simple, fast, quantitatively accurate, highly sensitive, etc. It has been widely used internationally and has been included in the pharmacopeias and biological product regulations of countries such as the United States, Europe, and China, and is used for the pyrogen test of drugs and biological products.
4. Removal of bacterial endotoxins
Due to the significant harm caused by endotoxins, the FDA and other regulations have clearly stated requirements for their control and reduction to safe levels. Aseptic operation and the cleanliness of the operating area are also effective means of controlling exogenous endotoxin contamination. However, for endogenous endotoxin contamination that already exists in the sample itself, even if exogenous endotoxins are effectively controlled, there is still a risk of exceeding the endotoxin limit in the final product. Therefore, it is necessary to find effective methods from the perspective of the sample preparation process and the characteristics of endotoxins, to reduce endotoxins in the sample itself and meet the process requirements, thus improving product safety.
4.1 Hydrophobic layer washing method
Generally, the hydrophobicity of endotoxins is much greater than that of the target substance. Therefore, in hydrophobic chromatography, a high-salt buffer is needed to balance the sample. Endotoxins aggregate under high salt conditions and do not bind to the hydrophobic medium, thus they are directly removed during sample loading. Alternatively, target samples can be loaded in flow-through mode at a certain salt concentration so that the target protein does not bind to the column packing material, while endotoxin molecules can bind to it, achieving separation between the two.
4.2 Ion exchange method
For anion exchange chromatography, endotoxins carry a negative charge at pH>2 and bind strongly to anionic exchange media such as Q or DEAE. Target proteins can be purified by flowing through an anion exchange column to remove endotoxins. Alternatively, target proteins and endotoxins can be bound to the chromatography medium simultaneously, and since endotoxins have a stronger binding capacity than proteins, target proteins can be eluted first, and then endotoxins can be washed out using a high salt buffer or NaOH. For cation exchange chromatography, endotoxins still carry a negative charge at pH 4.0 and cannot bind to the medium, so they flow out of the column with the mobile phase. Target proteins can bind to cation exchange media, making cation exchange chromatography also effective for removing endotoxins. In addition, the use of surfactants such as Triton X-114 can prevent endotoxins from binding to both anionic and cationic columns.
4.3 Gel filtration chromatography
This method can separate large molecular weight endotoxin molecules from target proteins based on the characteristic that endotoxins can form non-polar and ionic interactions with water-soluble solutions to form aggregates with a size of 1,000 kDa, which differs significantly from most biological protein molecules. However, its processing volume is small and the time is long.
4.4 Tangential flow filtration (TFF) technology
TFF can remove endotoxins. Endotoxins can form aggregate structures at different degrees and with different sizes in various aqueous solutions, with small aggregates having a molecular weight of 10-20 kDa and large ones up to 1,000 kDa. TFF can trap endotoxin molecules smaller than the pore size of the membrane while allowing the target sample to pass through the membrane pores, achieving the removal of endotoxins from the sample. Factors affecting endotoxin removal in protein solutions mainly include the size distribution of target molecules, the interaction between endotoxins and target molecules, the concentration of target proteins, and whether appropriate detergents are added.
4.5 Affinity chromatography
The use of affinity chromatography, particularly immunoadsorption agents, has made the purification of large biomolecules simple in production. The principle of immunoadsorption is based on the specific interaction between antigens and antibodies. Target proteins are used as antigens, and monoclonal antibodies produced by hybridoma technology are coupled to the medium to adsorb the target protein. Due to the high specificity of the interaction, only the target protein is theoretically adsorbed on the medium, and all endotoxins penetrate through. After elution, a highly-pure apyrogenic product can be obtained. In practice, due to the non-specific adsorption of the medium itself, a small number of impurities and endotoxins are still adsorbed, but the endotoxin content is very low. In interferon (IFN) production, the endotoxin content in the eluate is generally 0.25 EU/mL.
4.6 Using specific adsorption media
The endotoxin substrate LAL or polymyxin B (PMB) is coupled to the chromatographic medium to specifically adsorb endotoxins while proteins are not adsorbed and can flow out with the mobile phase. The collected protein can be used for further processing. This method has a good effect on endotoxin removal, but there is a certain protein loss.
In conclusion, there is currently no universal solution for removing endotoxins in biopharmaceutical processes. Especially for samples with particularly high endotoxin content, when a single removal method is not effective, it is necessary to consider a combination of different methods based on the characteristics of different biological products and processes to reduce endotoxin impurities in the process and improve product safety, thereby meeting relevant regulatory requirements.