Bacterial Endotoxin Testing ppt by biosolutions.

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 1940’s 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
100L of sample + 100L 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 100L ------ 100L
2 ------ 100L 2 100L
 ------ 100L  100L
/2 ------ 100L /2 100L
/4 ------ 100L /4 100L

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/ƒ)
Where,
e = Sum of log of Endpoint Concentrations
ƒ = 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/ƒ)
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