Medicilon can provide clients with method development, validation and testing of microorganisms and bacterial endotoxins for APIs and different types of drug products.
Bacterial endotoxins are cell wall components of gram-negative bacteria that are released when the bacteria die or autolyse. Its lipopolysaccharide (LPS) amphoteric macromolecule component is highly thermogenic. When endotoxin enters the human blood through injection or other methods, it can cause different diseases. In severe cases, it can cause shock or even death (pyrogen reaction). Therefore, pharmaceutical companies need to control bacterial endotoxins in all aspects including raw materials, packaging materials, original solutions, semi-finished products and finished products.
The bacterial endotoxin test (BET) is a method that uses lysate reagent to detect or quantify bacterial endotoxins to determine whether the limit of bacterial endotoxins in the test product meets the regulations. Including gel method (limit test, semi-quantitative test), photometric method (turbidity method, chromogenic matrix method), any one of these methods can be used for testing. When the measurement results are controversial, the results of the gel method shall prevail unless otherwise specified.
Medicilon has established a mature process for endotoxin testing and has completed endotoxin testing, method development and validation for hundreds of new drugs and generic drugs worldwide. The project cycle is short, fast and accurate, and it is widely praised by clients.
1 Experimental Preparation: reagents, consumables and instruments
The relevant endotoxin detection reagents, consumables and equipment used by Medicilon have been strictly screened and purchased from suppliers. Relevant reagents and equipment that are recognized on the market meet the requirements, such as the endotoxin standards, lysate reagents and inspection water from Zhanjiang Andusi Biology.
2 Determine the bacterial endotoxin limit of pharmaceutical products (L)
The bacterial endotoxin limit of a product is generally calculated through the formula L=K/M. Among them, M is the maximum dosage of the test substance per kilogram of body weight per hour for humans. Please refer to the drug instructions or authoritative information for usage and dosage. Medicilon's professional team can also provide relevant technical guidance and help clients quickly and accurately formulate relevant limit standards based on pre-screening test results.
3 Calculate the Maximum Valid Dilution (MVD) or Minimum Valid Concentration (MVC) of the test product
4 Review the sensitivity of lysate reagent
5 Preliminary screening test for compatibility between drugs and limulus reagents
6 Interference test of test article and verification of interference test
The gel method uses the principle that bacterial endotoxins react with limulus reagent to form a gel to determine whether the endotoxin content in the test sample meets the regulations. The reaction process will be affected by many factors, and interference problems often exist. For example, interference comes from the main components, auxiliary agents, and even some impurities in the sample. Most interference can be eliminated by diluting the test sample with bacterial endotoxin test water. When some interference cannot be eliminated using only the dilution method, other methods can be used to eliminate the interference before conducting the test.
Based on literature and practical cases, Medicilon has compiled a complete set of various interference factors and corresponding elimination methods, which can quickly and accurately eliminate interference and conduct verification tests.
Summary table of interference factors and elimination methods
| Interference Factors | Elimination Methods |
| pH interference | Adjust pH value to 6.0~8.0 |
| Interference caused by insufficient concentration of divalent cations | Add appropriate amount of Ca2+ and Mg2+ ion buffer solution |
| Interference caused by endotoxin accumulation | Vortex regularly, vortex before preparation |
| Interference from high concentrations of salt or sugar | Dilute or use appropriate ultrafiltration equipment |
| Liposome interference | Add endotoxin dispersant |
| Interference with endotoxin adsorption | Dilution tubes, reaction tubes, and graduated pipettes made of borosilicate glass are preferred, and polystyrene should be used for disposable utensils |
| Interference from surfactants | The test product is diluted with physiological saline. Physiological saline can salinize the test product, thereby reducing its surface activity. |
| Interference caused by enzymes and enzyme inhibitors | Heat the test sample appropriately to inactivate the enzyme |
| Dextran interference | Use an anti-solubilization lysate |
7 Daily endotoxin limit test of samples.
8 Suspicious result handling proceduresThe analyst first reviews the experimental project: standards, operations, records and calculations, instrument status, standard materials, reagent quality, other abnormal phenomena (whether there is possible contamination, environment, equipment, etc.)Review by the reviewer: If an abnormality is found in the inspection process of the inspector, the reviewer will repeat the experiment with the correct process to replace the original experimental results.If no abnormalities are found in the inspection process of the inspector, the reviewer can repeat the experiment in order to determine whether the unqualified results are caused by testing or analysis errors.Quantitative testing provides data referenceThe use of different lysate reagents
Background:In the liposome excipient process R&D and production project, due to limitations, the customer has never successfully developed a method for endotoxin detection. As mentioned above, some interference factors due to the introduction of the process or the existence of the product itself in endotoxin detection cannot be eliminated even when the product is diluted to the maximum effective dilution volume . Inhibition of the reaction between endotoxin and lysate reagent causes the positive control group of the test product to test negative.
Medicilon's microbiology team developed a product pre-treatment method that can eliminate many interfering factors in a short period of time based on process steps and previous experience by eliminating a complete set of relevant interfering factors.
1. Liposomes have poor water solubility
The amphiphilic nature of lipopolysaccharide will give it an affinity for some products with hydrophobic groups. The combination of lipopolysaccharide with these products will affect their size and shape, thus affecting their activity, causing interference in endotoxin tests and causing inhibition. By adding an appropriate amount of endotoxin dispersant to separate the endotoxin in the product, the activity of the endotoxin can be restored.
2. Chelating agent interference
The reagents introduced in the process contain chelating agents, which will chelate the divalent cations (such as Mg2+ ions) in the limulus reagent, resulting in insufficient cations and inhibition. At the same time, an appropriate amount of Mg2+ ion buffer is added to eliminate the interference caused by insufficient divalent cations caused by the related chelating agents introduced in the process, so that the endotoxin can normally activate the enzyme reaction of the limulus reagent.
In summary, the interference from various factors was eliminated, and the interference experiment was successfully verified and the limit inspection was completed at the maximum allowable dilution concentration. This helped the clients to achieve rapid delivery within a short cycle.
Reference:
Zhanjiang A&C Biological LTD.
Chinese Pharmacopoeia 2020 Edition Four General Principles 1143 Bacterial endotoxin test method
Chinese Pharmacopoeia 2020 Edition Four General Principles 9251 Guidelines for the application of bacterial endotoxin testing methods
Bacterial Endotoxin Test Method (Gel Method) Experimental Procedures
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