![Calculation Of Geometric Mean
Formula :
GM end point Concentration = antilog(e/)
Where,
e = Sum of log of Endpoint Concentrations
= Number of Replicates
Calculation :
GM = log (0.125) + log (0.125) + log (0.0625) + log (0.0625)
4
= Anti[(-0.9030) + (-0.9030) + (-1.2041)+ (-1.2041)]
4
= Antilog [(-1.0536)]
= 0.0883 EU/mL
Label Sensitivity is Confirmed if GM endpoint is between 2 and /2](https://image.slidesharecdn.com/betbasicpresentation-250330084023-5155ef48/85/Bacterial-Endotoxin-Testing_Basic_Presentation-ppt-26-320.jpg)
![Endotoxin Limit ( EL)
The limit formula for radio pharmaceuticals is
175/V except for intrathecally administered
products. 14/V for intrathecal drugs.
V equals the maximum recommended dose, in mL,
at the expiration date or time.
For drugs administered on a per Square Meter of
Body Surface:
2.5 EU/ [ ( dose * 1.8 sq.. m.)/ 70 Kg]](https://image.slidesharecdn.com/betbasicpresentation-250330084023-5155ef48/85/Bacterial-Endotoxin-Testing_Basic_Presentation-ppt-30-320.jpg)
![Geometric Mean Calculation
GM end point Concentration = antilog(e/)
GM in Water = log (0.125) + log (0.125) + log (0.125) + log( 0.125)
4
= Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)]
4
= Antilog [(-0.9030)]
= 0.125 EU/mL
GM in Product = log (0.125) + log (0.125) + log (0.125) + log( 0.125)
4
= Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)]
4
= Antilog [(-0.9030)]
= 0.125 EU/mL](https://image.slidesharecdn.com/betbasicpresentation-250330084023-5155ef48/85/Bacterial-Endotoxin-Testing_Basic_Presentation-ppt-43-320.jpg)
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Bacterial Endotoxin Testing_Basic_Presentation.ppt
- 1. Introduction to Bacterial Endotoxins Test Bio Solutions
- 2. Bacterial Endotoxins Test (BET) LAL Limulus Amebocyte Lysate (Limulus polyphemus) TAL Tachypleus Amebocyte Lysate (USP 29 only) (Tachypleus tridentatus, Tachypleus gigas) CAL Carcinoscropius Amebocyte Lysate (Carcinoscropius rotundicauda)
- 7. Bacterial Endotoxins Ultrastructure Of Gram ve Bacterial Cell Wall Lipopolysaccharide
- 8. Endotoxicity
- 9. Rabbit Pyrogen test From 1940s Rabbit Pyrogen test was method of choice for detection of pyrogen. This Rabbit pyrogen test was incorporated into USP XII in 1942.
- 10. Accidental Discovery Of LAL Dr. Frederick Bang Dr.Jack Levin
- 12. Reference Standard Endotoxin In 1974 first Reference Standard endotoxin was prepared Dr. J. A. Rudbach from Escherichia coli 0113:H110:K. In 1976 Lot EC 1 was prepared.
- 13. Currently EC - 6 (FDA RSE) or Lot G (USP RSE) Potency 10,000 EU/ vial
- 14. In 1980 USP XX included BET using LAL In 1987 the FDA published The Guideline on validation of the LAL test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices.
- 15. Biochemistry Of LAL Endotoxin Factor C Factor C (active) Factor B Factor B (active) Proclotting Enzyme Clotting Enzyme Coagulogen Coagulin
- 16. Procedure For LAL Test 100L of sample + 100L of LAL reagent 10X75 mm tubes 37 1 C 60 2 min look for Gel formation after inverting tube by 180
- 17. Reagents Lyophilized Wako LAL reagent, Sensitivity ( EU/ml) Control Standard Endotoxin (CSE), for Control Curve and PPC* LAL Reagent Water (LRW), Diluent
- 18. Accessories Vortex Mixer Heating Block Depyrogenated Glass Test Tubes for Assay (10X75mm) Depyrogenated Glass Test Tubes for Dilutions Micro Pipette Sterile Micropipette Tips Timer
- 19. Initial Quality Control Analyst Qualification Verification of Testing accessories Verification of label claim sensitivity
- 20. As per USP : New Batch of LAL Reagent Any Change in Experimental Conditions Four Standards Concentrations in Quadruplicates with Negative Controls
- 21. Reconstitute Control Standard Endotoxin (CSE) referring to CoA. Dilute the CSE to 2, , /2 and /4 EU/mL concentrations. Where is sensitivity of LAL reagent
- 22. Tubes LRW CSE LAL Blank 100L ------ 100L 2 ------ 100L 2 100L ------ 100L 100L /2 ------ 100L /2 100L /4 ------ 100L /4 100L
- 23. Results Of Control Curve Valid I II III Blank 2 (0.25 EU/ml) + + + + + + + + + + + + (0.125 EU/mL) + + + + + + + + /2(0.06 EU/mL) + + + + /4(0.03 EU/mL)
- 24. Results Of Control Curve Invalid I II III Blank + + 2 (0.25 EU/mL) + + + + + + + + (0.125 EU/mL) + + + + + + + + /2(0.06 EU/mL) + + + + + + + + /4(0.03 EU/mL) + + + + + ++ +
- 25. Results Of Control Curve Tubes Blank 2 /2 /4 End Point 1 + + 0.125EU/mL 2 + + + 0.0625EU/ml 3 + + 0.125EU/ml 4 + + + 0.0625EU/mL
- 26. Calculation Of Geometric Mean Formula : GM end point Concentration = antilog(e/) Where, e = Sum of log of Endpoint Concentrations = Number of Replicates Calculation : GM = log (0.125) + log (0.125) + log (0.0625) + log (0.0625) 4 = Anti[(-0.9030) + (-0.9030) + (-1.2041)+ (-1.2041)] 4 = Antilog [(-1.0536)] = 0.0883 EU/mL Label Sensitivity is Confirmed if GM endpoint is between 2 and /2
- 27. Endotoxin Limit ( EL) FDA established Endotoxin Limits based on Formula EL = K ( Tolerance limit ) M (maximum dose/ Kg/Hr) EL represents the maximum safe amount of endotoxin.
- 28. Endotoxin Limit ( EL) K = 5 EU/Kg Body weight for parenteral drugs except those administered intrathecally. 0.2 EU/kg for intrathecal drugs
- 29. Endotoxin Limit ( EL) M = Maximum dose administered to a patient per kg Body weight, per hour (no heroic dose).
- 30. Endotoxin Limit ( EL) The limit formula for radio pharmaceuticals is 175/V except for intrathecally administered products. 14/V for intrathecal drugs. V equals the maximum recommended dose, in mL, at the expiration date or time. For drugs administered on a per Square Meter of Body Surface: 2.5 EU/ [ ( dose * 1.8 sq.. m.)/ 70 Kg]
- 31. Product Testing Negative Product Control (NPC) - Sample + LAL Positive Product Control (PPC) - Sample + CSE (2) + LAL Negative Water Control (NWC) - LRW + LAL Positive Water Control (PWC) - LRW + CSE (2) + LAL
- 32. Interference Significant difference between the end points of positive water control and positive product control using standard endotoxin.
- 33. Interference Sub optimal pH concentration Endotoxin modification Container effects Unbalanced cation levels Protein or Enzyme modification
- 34. Maximum Valid Dilution (MVD) (Potency of the product) x (Endotoxin Limit) MVD = __________________________________ Potency of product = Concentration of the product in units/mL Endotoxin Limit = K/M lambda ( ) = Lysate label claim sensitivity
- 35. Example Drug : Cefotaxime Sodium Sterile 500mg/2mL Endotoxin Limit : NMT 0.2 EU/mg Lysate sensitivity ( ) : = 0.125 EU/mL MVD = Conc. of Drug (potency) x E.L = 250mg/ mL X 0.2 EU/mg 0.125EU/mL MVD = 400
- 36. Minimum Valid Concentration (MVC) MVC = / E.L.
- 37. Example Drug : Cefotaxime Sodium Sterile Endotoxin Limit : NMT 0.2 EU/mg Lysate sensitivity : 0.125 EU/mL MVC = / E.L. = 0.125 EU/mL 0.2EU/mg = 0.625mg/ mL
- 38. Product Validation The validation for LAL compatibility of a drug product is a test condition where an endotoxin standard is detected with the same efficiency in a test sample as it is in LRW.
- 39. Product Validation Phase I: Preliminary Screening Determines the Non Interfering Dilution / Concentration of the product which is used for actual validation. Phase II: Product Validation
- 40. Phase I: Preliminary Screening Prepare dilutions of product up to MVD such as, MVD / 16, MVD / 8, MVD / 4, MVD / 2, MVD Run NPC AND PPC Least DILUTION / highest CONC. of product for which PPC is Positive and NPC is Negative is the Non Interfering Dilution (NID)/ Non Interfering Concentration (NIC).
- 41. Screening For Non Interfering Dilution / Concentration Sample Dilutions 1:50 1:100 1:200 1:400 Test sample Concentration 5 2.5 1.25 0.625 (mg/mL) Unspiked Spiked (2 ) + + + + + + Non Interfering Dilution = 1:100 Non Interfering Concentration = 2.5mg /mL
- 42. Test Sample LRW Sample CSE LAL Results Negative water control 100 L 100 L 2 (0.25EU/mL) 100 L 2 100 L + + (0.125EU/mL) 100 L 100 L + + /2(0.06EU/mL) 100 L /2 100 L /4(0.03EU/mL) 100 L /4 100 L Negative product control 50 L 50 L (1:200) 100 L Positive product control 2 50 L (1:200) 50 L 4 100 L + + + + Positive product control 50 L (1:200) 50 L 2 100 L + + + + Positive product control /2 50 L (1:200) 50 L 100 L Positive product control /4 50 L (1:200) 50 L /2 100 L
- 43. Geometric Mean Calculation GM end point Concentration = antilog(e/) GM in Water = log (0.125) + log (0.125) + log (0.125) + log( 0.125) 4 = Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)] 4 = Antilog [(-0.9030)] = 0.125 EU/mL GM in Product = log (0.125) + log (0.125) + log (0.125) + log( 0.125) 4 = Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)] 4 = Antilog [(-0.9030)] = 0.125 EU/mL
- 44. If endpoint of CSE in product is within one two fold of endpoint in water Product is Validated
- 45. Gel-clot Limits Test By 50 50 Method LRW CSE (4) Sample (MVD/2) LAL NWC 100 L ------ ------ 100 L PWC 50 L 50 L ------ 100 L NPC 50 L ------ 50 L 100 L PPC ------ 50 L 50 L 100 L
- 46. Expected Results Pass Interferes Fails Repeat NWC PWC + + + + + + + + NPC + + + PPC + + + + + +
- 47. Applications Large Volume Parenterals (LVPs) Multiple - ingredient drugs Small Volume Parenterals (SVPs) Radiopharmaceuticals Biologicals Water system validation Validation of Dry heat Sterilizer Medical devices