Endotoxin Test and Sterility Test

ENDOTOXIN TEST AND STERILITY TEST TOOLS AND MATERIALS • Sterile pyrogen-free bottles • Incubator with a temperature range of 36-38°C • LAF units • Pyrogen-free measuring pipette or pyrogen-free syringe • Test tube rack • 3 vials of Pyrotel Limulus Amebocyte Lysate (LAL) 0.125 EU/ml orother appropriate sensitivities • 1 control vial of Endotoxin Standard 5000 EU/vial • LAL reagent water EXPERIMENT PROCEDURE General rules • Glass containers used for testing must be washed thoroughly, soap residue must be completely removed. Glass containers must also be pyrogen-free by heating the glass container in a sterilizer oven at 200ºC for 1 hour. • To prevent microbial contamination, all testing stages must be carried out aseptically. • The temperature during the reaction must be between 36-38ºC. • The pH of the solution should be between pH 5-7. If necessary, adjust the pH with 0.1 N HCl, 0.1 N NaOH, or an appropriate buffer solution. • Reaction time is 1 hour. • Each test must be accompanied by a negative control and a positive control. • When testing, efforts must be made to ensure that the area is free from vibration. • Store pyrotel, CSE vial, or endotoxin stock solution according to instructions from the supplier. LAL storage conditions and Endotoxin Control Standards • Store Pyrotel Limulus Amebocyte Lysate in the refrigerator (temperature -20 to °C). • Store Control Standard Endotoxin in the refrigerator (temperature 2-8 °C). • Endotoxin stock solution that has been dilted with LAL reagent water can be stored at a temperature of 2-8ºC for 4 weeks. Label the manufacturing date and expiration date on the endotoxin stock solution container. • Pyrotel Limulus Amebocyte Lysate and Control Standard Endotoxin stored in the refrigerator will have good activity until the expiry date. LAL Test on Water For Injection i. Procedure for making Endotoxin stock solution • Dilute the Control Standard Endotoxin vial (5000 EU/vial) with 5 ml LAL reagent water to obtain a concentration of 1000 EU/ml (solution A). • Pipette 1.0 ml of solution A and dilute with LAL reagent water to 10 ml (solution B). • Pipette 1.0 ml of solution B and dilute with LAL reagent water to 10 ml (solution C). • Pipette 0.25 ml of Solution C and dilute with LAL reagent water to 10 ml (solution D= Stock solution 0.25 EU/ml). ii. Work procedures • Remove 3 tubes of Pyrotel Limulus Amebocyte Lysate 0.125 EU/mL, LAL reagent water and endotoxin stock solution from the refrigerator. • Leave for 30 minutes until the temperature is the same as room temperature (25- 30°C). • Add to the pyrotel tube 0.125 EU/ml each:- 0.2 ml WFI sample - 0.2 ml LAL reagent water (as negative control) - 0.2 ml endotoxin stock solution 0.25 EU/ml (as positive control)aseptically under LAF. • Shake the tube for 20 to 30 seconds to mix homogeneously. • Place the pyrotel tube in an incubator or water bath at a temperature of 37 ± 1°C. • Incubate for 1 hour. • Observe the tube containing the sample, positive and negative controls • Record the results Interpretation of Results • If a gel forms in the tube containing the sample and if the vial is rotated 180° and the gel remains at the bottom of the vial then the LAL test result is declared positive. • LAL test results are declared negative if it does not form slowly.The gel and liquid flow when the vial is rotated to 180°. • Positive controls must provide positive results for LAL • Negative controls must give negative results for LAL. • Record LAL test results. K3L Aspects (Health, Work Safety and Environment) Discard used pyrotell and endotoxin standard controls as pharmaceutical waste