The importance of testing for bacterial endotoxin concentration

Posted by TS. Nguyen Dac Tu, Team Leader of Product Quality Evaluation of High Technology Center and MSc. Nguyen Thi Mai, Bacterial Endotoxin Test Specialist, High Technology Center

Bacterial endotoxin when entering the blood or cerebrospinal fluid in high concentrations will cause fever, even septic shock, which can lead to death. Endotoxins are very common in the environment, cannot be removed by conventional sterilization methods, so they are easy to contaminate products such as intravenous drugs, vaccines, medical equipment, biological products such as cells, etc. Stem cells, immune cells, cell products... There needs to be a plan to control and determine the concentration of endotoxins in these products to ensure safety when used for patients.

Bacterial endotoxins are a form of pyrogens, which are essentially lipopolysaccharides (LPS) derived from the outer wall of gram-negative bacteria. LPS is composed of 3 parts: the outermost -0 antigen (0- antigen), the core in polysaccharides, and the Lipid A part containing fatty acids, which is the main part that determines the virulence of the endotoxin. This structure is essential for maintaining cell wall integrity and survival of gram-negative bacteria. These bacteria release to the environment small amounts of endotoxins as products of their metabolism, and large amounts of endotoxins are released when cell walls are damaged or when gram-negative bacteria are damaged. dead or dissolved.

Endotoxins are a potent stimulant of the innate immune system. When amounts of endotoxins enter the systemic circulation that exceed the threshold allowed by the US FAD, they trigger an immune response, usually inflammation, that produces cytokines such as interleukin 1 and tumor necrosis factor. These secretory signals attract neutrophils to migrate, activating phagocytosis. When a patient has a weakened immune system, has an underlying disease, or is infected with a large amount of endotoxin, the clinical symptoms of septic shock include fever, decreased blood pressure, decreased circulation in the organs, causing damage to body tissues such as heart, lungs, liver, kidneys,.. the immune system secretes enzymes to destroy cells, metabolic disorders due to lack of cellular oxygen, damage to internal cells membranes, increased vascular permeability and possibly death.

Because of their bacterial origin, endotoxins are very common in the air, water, and soil. In addition to its thermal stability (thermogenic) and ability to escape from 0.2 μm membranes, conventional decontamination methods cannot remove endotoxins. This means that a product that is certified as sterile may still have endotoxins in it. Because of the popularity in the environment, the ability to withstand heat and withstand sterilization methods of pharmaceutical manufacturing processes, injectable drugs, vaccines, medical devices and biological products such as stem cells. , immune cells, cell secretions... as a result, the product is highly susceptible to bacterial endotoxin contamination. That is also the reason that every step in the production of pharmaceuticals or biological products must carry out endotoxin control at appropriate times, at least the final product before use in humans.

100% of injected pharmaceutical products, implanted medical devices, and end products of biological manufacturing processes that come into contact with blood or CSF must be tested for endotoxins and concentrations within within the limits recommended by FDA. These include:
Drugs for injection (including distilled water for injection) Vaccines Medical devices that come into contact with blood vessels Biological products that are administered through blood vessels or cerebrospinal fluid such as stem cells, immune cells, cell products, protein solutions.

Endotoxin limits are usually specified in separate monographs. The product is qualified if the amount of endotoxin present in the product is below the limit recommended by the US FDA. The endotoxin limit was calculated on a human dose basis using the formula: EL=K/M. where K is the endotoxin threshold calculated for 1 kg of human body weight for 1 hour tolerable without a toxic reaction. This dose is understood as the maximum dose. If a product can be administered by both intravenous and cerebrospinal fluid exposure, the endotoxin limit is calculated using the K value of the lower endotoxin limit route, so that when administered Maximum product safety for patients. Regulated endotoxin limits are detailed in the table:

The processing and culturing of stem cells and immune cells as well as the production of cell products for clinical use is a lengthy process in which these products have been exposed to a wide variety of pathogens. consumables such as bottles, culture dishes, pipettes, membranes, and chemicals such as culture media, buffers, etc. These can cause an increase in bacterial endotoxin concentrations in the final product before production. use for the patient. Without measures to control this problem, the cell product used is not only ineffective, but also dangerous to the patient because of the amount of endotoxin contained in it. This is the reason that international standards for cell therapy such as ISCT, FACT, and AABB classify endotoxin testing as mandatory testing for cellular products prior to their therapeutic use. . Based on FDA regulations on bacterial endotoxin limits for each infusion line and test results to determine endotoxin concentration, doctors will have enough basis to calculate the appropriate infusion time to ensure ensure patient safety.
According to the provisions of international standards for cell therapy such as ISCT, FACT, AABB, endotoxin determination test is classified as a mandatory test for cell products before use for patient.
In order to ensure the highest safety for customers according to international standards, the High-Tech Center, Vinmec Times City Hospital has developed and performed a test to determine the concentration of bacterial endotoxins. for stem cell and immune cell products based on an FDA-approved method.

References:
USP, Chapter <85>, Bacterial endotoxin test, 161-167, U.S.Pharmacopeial convention, Rockville, MD,2015. USP 39, Chapter <161>, Medical devices- bacterial endotoxin and pyrogen tets, 219-222 , U>S>Pharmacopeial convention, Rockville,MD, 2016. FDA, guideline on validation of the limulus amebocyte lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices, 1987. Endotoxin limits for parenteral drug products, Bet white paper vol.1 no.2, 2017 The Code of Federal Regulations, 21 CFR 211,167 Vietnamese Pharmacopoeia, Endotoxin Testing Appendix 13.2 Comparison of Endotoxin Testing Methods for Pharmaceutical Products. Timothy J. Joiner Paul F. Kraus Thomas C. Kupiec, PhD Analytical Research Laboratories, Oklahoma City, Oklahoma. International Journal of Pharmaceutical Compounding Vol. 6 No. 6 November/December 2002