Test method for determination of 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

With the recent massive development of cell therapy application centers, many facilities do not meet compliance with product quality control procedures, including standards for endotoxins. This will pose many potential risks to customers using medical services at these facilities.
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 Technology Center, Vinmec Times City Hospital has developed and performed a test to determine the concentration of bacterial endotoxins for each patient. stem cell and immune cell products based on an FDA-approved method.

1.Bacterial endotoxin test Bacterial endotoxin test is used to detect or quantify bacterial endotoxin present in the test sample. The method of using lysate reagent is the amoeba cell lysates found in the blood of the sea buckthorn, Limulus polyphemus or Tachypleus tridentatus. There are currently 3 methods to perform the test:
Gelation method based on gel formation. When the sample to be tested is added to a tube containing a reagent, a gel is formed in the presence of endotoxins. Turbidimetric method, based on the change in turbidity of lysate reagent during gel formation. The kinetic colorimetric method is based on the color change of the color-peptide complex. Depending on the conditions of each laboratory and the nature of the sample, one of the appropriate techniques will be used to perform the test and the test will use endotoxin-free test equipment and instruments. In the event of doubt or dispute, the final conclusion will be based on the gelation method unless otherwise indicated.

2. Gelation method This is a method that allows to detect or determine the amount of endotoxin on the gelation of lysate reagent in the presence of endotoxin. Using a blood-clotting protein found in sea buckthorn. This protein is activated in the presence of endotoxins. The gelation reaction consists of a series of enzyme activation steps in Limulus Amebocyte Lysate. The test is performed by adding 0.1 mL of test sample to a prepared test tube of 0.1 mL of LAL reagent solution. The mixture was mixed well and incubated at 37°C for 60 min. Read the results after this incubation period, positive reaction if the gel formed does not flow when gently inverting the test tube (180°), negative reaction when no gel is formed or the gel is not strong enough. The limitation of the gel-clot method is that the test time is long and the preparation is high, and the error due to manipulation is quite high. For qualitative results only. The exact concentration in the test sample could not be determined.
3. Turbidity method Also known as semi-quantitative test. The turbidimetric method is similar to the kinetic chromogenic method in terms of method and calculation of the amount of endotoxin present in the specimen. This test determines the amount of endotoxin present in the test sample by performing a reaction that approaches a breakpoint during gelation. It is an intermediate method between gelation and kinetic colorimetry except for the last step. Steps involved in cleavage of a coagulation protein via an activated inducer enzyme. The cleavage products conjugate with each other as a result of ionic interactions that occur after the cleavage, the reaction mixture becomes turbid. The turbidimetric method determines endotoxin concentration in two ways: kinetic and endpoint. Both methods require the construction of a standard curve. The endotoxin concentration in the sample was determined by measuring the optical density against the standard curve, the time interval until the turbidity level of the lowest concentration of the standard curve was reached.

4. Colorimetric method Also known as kinetic colorimetric. The method was developed based on gelation and turbidimetric methods in 1977. Endotoxins in test samples were determined based on the colorant released from a chromogenic substrate as a result of the reaction between endotoxin with lysate reagent. There are two measurement techniques: stop colorimetry and kinetic colorimetry. Kinetic colorimetry is based on the relationship between concentration and the time it takes for the reaction mixture to reach absorbance. This method also needs to build a standard curve, to build the curve requires a minimum of 3 concentrations. The endotoxin concentration in the test sample is colored against the standard curve. This is the most commonly used method today because of its convenience and high sensitivity.

5. Some points to note in the endotoxin test The endotoxin test is not suitable for some types of samples such as emulsions, suspensions,... some substances may give positive reactions. positive or false negative with lysate reagents such as blood products, polynucleotides, preparations containing heavy metals, surfactants, high ion concentrations. When in the sample there is an inhibitor or enhancer of LAL activity. The most effective way to pass is sample dilution, heat treatment, pH neutralization....
Endotoxin determination method by Charles River Endosafe system approved by FDA, USA is being Using at Vinmec Hospital
Based on the colorimetric method, Charles River, USA has improved the kinetic colorimetric method into a card form, testing endotoxins within 30 minutes, suitable for cell therapies samples that need to be transplanted to the patient shortly after processing or harvesting. The advantage of time is achieved by the standard curve built and built into the endosafe machine system.

Vinmec Times City International General Hospital is performing endotoxin testing by kinetic colorimetric method using the Endosafe endotoxin testing system of Charles River, USA. This is the most commonly used method in the world today because of many advantages:
Very high sensitivity Specific for endotoxins For quick results in just 30 minutes, meeting the needs of results folding fruit. The card uses FDA certification, the endotoxin detection tag range has a high sensitivity of 0.5 - 0.005 EU/ml. The High Technology Center, Vinmec Hospital is also one of two Vietnamese facilities to receive ISO 15189 certificate for the test to determine bacterial endotoxin concentration using the Endosafe system of Charles River. ISO 15189:2012 is a set of standards that specifies quality requirements for internationally recognized medical laboratories. The set of standards includes 15 management requirements and 10 technical requirements for quality assurance in testing activities such as: capacity and skills of testing staff; control environmental conditions; test equipment control; pre-test preparation; control the testing process...The application of ISO 15189 standard will help ensure accurate, timely and reliable delivery of test results, serving as a basis for promoting the recognition of results. testing between facilities for examination and treatment of patients. With the use of testing methods approved by FDA and ISO 15189:2012 certificate, Vinmec is committed to bringing the safest cell therapy treatment process to patients.

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 test, 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