Bacterial Endotoxins Test

(BET)
An Introduction

Ravinder Singh
(Technical Executive)
Charles River Laboratories India Pvt. Limited
Chandigarh
 Bacterial Endotoxins Test ( BET )

• Bacterial Endotoxins Test ( BET ) is the most sensitive

and specific test available to detect and measure the
presence of Endotoxin, a fever producing byproduct
of Gram Negative Bacteria, commonly known as
Pyrogen.

• FDA and USP guidelines require final product testing

of all Parenterals and medical devices.
 What are Endotoxins ?
• Chemically Endotoxins are Lipopolysaccharide (LPS)
complex

• Part of the outer membrane of the cell wall of Gram-

negative bacteria.

• Released during the growth of Gram Negative

bacteria and also after its death
Ultra structure of Gram Negative
Bacteria Cell Wall
General Structure of Lipopolysaccharide
Biochemical Response of LPS

Endotoxin

Macrophages / Monocytes / Neutrophills

IL-1, IL-6 TNF

Pyrogenicity Coagulation
Hypotension Inflammation
 Rabbit Pyrogen Test (RPT)
• In the Rabbit Pyrogen Test three healthy rabbits are injected
drug sample by the intravenous route and rectal temperature
of the rabbits are monitored every half an hour for three
hours

• If more than 0.6 degree rise in temperature is measured

individually, then the product is considered to be pyrogenic

• Approved by FDA in 1941

• Pyrogen Test became an official Quality Control test in 1942

in USP 12th edition
 Limitations of Rabbit Pyrogen Test

• In vivo model (Rabbit to rabbit variations)

• Sensitivity
• Specificity
• Time Consuming
 Discovery of LAL
• Dr. Frederick Bang (1960’s)
Pathologist at Woods Hole Oceanographic Institute

• Dr. Jack Levin

Hematologist at Johns Hopkins, developed LAL
reagent

• Dr. James Cooper (1969)

Pharmacist and Graduate student at Johns Hopkins
University
Limulus polyphemus (Horseshoe Crab)
Limulus polyphemus (Horseshoe Crab)
Blood Extraction
Limulus Amebocyte Cells Extraction
 Biochemistry of LAL
Endotoxin

Factor C Factor C (Active)

Factor B Factor B (Active)

Proclotting Enzyme Clotting Enzyme

Coagulogen Coagulin
 Other Lysate

• LAL : Limulus Amebocyte Lysate

(Limulus polyphemus)

• TAL : Tachyphleus Amebocyte Lystae

(Tachyphleus tridentateus,
Tachyphleus gigas)

• CAL : Carcinoscorpius Amebocyte Lysate

(Carcinoscorpius rotundicauda)
 FDA Approval
• In 1980, USP XX included BET using LAL

• 1n 1987 the FDA published The Guidelines on

validation of the LAL test for human and animal
Parenterals drugs, biological products, and medical
devices.
 LAL Methods
• Gel Clot Method

• Kinetic Methods
1. Turbidimetric
2. Chromogenic

• Portable Testing System (PTS)

GEL CLOT METHOD
 LAL REAGENTS AND ACCESSORIES

REAGENTS:
 Licensed LAL Reagent
 Control Standard Endotoxins
 LAL Reagent Water

EQUIPMENTS:
 Dry Heating Block
 Vortex Mixer

ACCESSORIES:
 Endotoxin- free dilution tubes, pipettes, pipette tips
 Calibrated micropipette
 Procedure of Gel Clot Test

100µL of Sample + 100µL of LAL Reagent

(10x75mm tubes)

Incubate at 37+1° C
For 60+2 Minutes

Observe for Gel Formation by inverting

tube at 180 ۫

 + ve means Firm Gel

 − ve means Less than firm Gel
 Reading Gel Clot Test

Positive

Cloudy

Negative
 Initial Quality Control

• Analyst Qualification
• To verify Label Claim sensitivity Control Curve
• Verification for Accessories

As per USP

• New batch of LAL reagents

• Any change in Experimental Conditions
 Steps for Initial Quality Control
• Reconstitute CSE referring to Certificate of Analysis
(CoA)
• Dilute CSE to 2λ, λ, λ/2, λ/4 EU/mL (λ is Lysate label
claim sensitivity)
• i.e. If  = 0.125 EU/ml, then dilute CSE in range of,
2 = 2 X 0.125 = 0.25 EU/ml
 = 0.125 EU/ml
 /2 = 0.125 / 2 = 0.0625 EU/ml
 /4 = 0.125 / 4 = 0.03125 EU/mL

• Reconstitute LAL Reagent

 Control Curve

Test Tube No CSE LRW LAL Reagent

1, 2, 3, 4 NWC - 100µL 100µL

5, 6, 7, 8 2λ 100µL - 100µL

9, 10, 11, 12 λ 100µL - 100µL

13, 14, 15, 16 λ/2 100µL - 100µL

17, 18, 19, 20 λ/4 100µL - 100µL

 Results of Control Curve- VALID

Tubes I Result II Result III Result

NWC ---- ---- ----

2λ ++++ ++++ ++++

λ ++++ ---- ++++

λ /2 ---- ---- ++++

λ /4 ---- ---- ----

Comments Ideal Valid Valid

 Results of Control Curve - INVALID

Tubes I Result II Result III Result

NWC ---- ---- ++++

2λ ---- ++++ ++++

λ ---- ++++ ++++

λ /2 ---- ++++ ++++
λ /4 ---- ++++ ++++
Comments Invalid Invalid Invalid
Dilutions (?) CSE Accessories, LRW
Reason Storage(?)
Dilution (?)
(?)
 Calculation of Geometric Mean

GM = antilog ∑e / f
Where,
e = Log (endpoints)
f = Number of Replicates
 Example Of Control Curve

Tubes I 2 3 4

Blank    
2 + + + +
 + + + +
/2  +  +
/4    
0.125 0.0625 0.125 0.0625
End Point
EU / mL EU / mL EU / mL EU / mL
 Calculation of GM

GM = log (0.125) + log (0.125) + log (0.0625) + log (0.0625)

4
= Anti [(-0.9030) + (-9030) + (-1.2041)+ (-1.2041)]
4
= Antilog [(-1.0536)]
= 0.0883 EU/mL

• Label Claim is verified / Analyst is qualified if the GM of

the endpoints confirms the Label Claim sensitivity of
the reagent +/- one two fold dilution
 Product Testing

• Endotoxin Limit
• Maximum Valid Dilution / Minimum Valid
Concentration Calculations
• Interference
 Endotoxin Limits (EL)
K (Tolerance Limit) EU/Kg/Hr
Endotoxin Limit =
M (Maximum Dose/Kg/Hr)

• Endotoxin Limits list is published by Regulatory Authorities (USP,

IP, BP, EP)

• Maximum dose administered to a patient per kilo per hour (no

heroic dose). For this calculation, it is assumed that the average
person weight is 70Kg. If the pediatric dose is higher, it shall be
used in the calculations.
 Endotoxin Limit
• For formulations (usually anticancer products)
administered on a per square meter of body surface

• Endotoxin Limit = K
(M X 1.80 m2) / 70 Kg
where
K = 5 EU/ Kg / Hr
M = The maximum dose / Kg / Hour.
Endotoxin Tolerance for Parenteral Products
Parenteral Type Endotoxin Tolerance Limit (K)

Human or Veterinary Drugs and Biologics 5 EU/kg

Parenterals by intrathecal injection 0.2 EU/kg

Radiopharmaceuticals 175 EU/V +
Intrathecal radiopharmaceuticals 14 EU/V +
Large Volume Parenterals 0.5 EU/mL
Water for Injection 0.25 EU/mL
Medical devices by extraction 0.5 EU/mL up to 20 EU/device

Medical devices in intrathecal spaces 0.06 EU/mL up to 2.15 EU/device

+ maximum dose in volume, by mL. * recommended limit by Dr. Cooper, not the HBET.
 Interference
 Both USP BET and EP/BP require that
Interference testing be performed on products
before routine testing can be instituted in the
lab.
 Interference is any physical or chemical inhibition
or enhancement which can alter the recovery of
Endotoxins
 Interference is caused by
 sample interaction with the LAL reagent
 sample interaction with endotoxin
 Types of Interferences

Inhibition : Endotoxin recovery

less than expected

Enhancement : Endotoxin recovery is

more than expected
 Interference Mechanisms
 pH Range (6.5 - 8.0)

 Endotoxin Depletion - Aggregation & Adsorption of CSE

 Cations (Na+, Ca 2+, Mg 2+)

 Container Effects

 Enzyme or Protein modifications

 Non-specific LAL activations [(1,3) ß-D- Glucan]

 Solution to Interference
Problem Solution
pH • Simple Dilution
• Addition of dilute acid/base
Endotoxin Depletion • Simple dilution
• Vortexing,
• Endotoxin dispersing agents
Cation Depletion • Dilute Chelating effect,
• Replace Ca2+ and Mg2+ deficits,

Container Effects • Avoid Polypropylene

Enzyme/ Protein • Dilution,

modification • Heat inactivation (70-80°C for 5 min)
Nonspecific • Avoid use of cellulose filters
activations • Use of Glucan Blocking buffer
 Maximum Valid Dilution (MVD)

MVD = Endotoxin Limit X Potency

λ
Potency = Conc. of products in Units/mL or mg/mL

Endotoxin Limit = K/M

Lambda (λ) = Lysate Label Claim Sensitivity EU/mL

MVD is a dilution factor.

 Example of MVD Calculation
Drug : Cefotaxime Sodium Inj.
Endotoxin Limit : NMT 0.2 EU/mg
Lysate Sensitivity : 0.125 EU/mL
Potency : 500mg/mL

Calculations:
MVD = Endotoxin Limit X Potency
λ
= 0.2 EU/mg X 500 mg/mL
0.125 EU/mL
MVD = 800

* MVD has no Units

 Minimum Valid Concentration (MVC)
Minimum Valid Concentration = Lysate Sensitivity (λ)
Endotoxin Limit
Example:
If, Drug : Cefotaxime Sodium Inj.
Endotoxin Limit : NMT 0.2 EU/mg
Lysate Sensitivity : 0.125 EU/mL
Potency : 500mg/mL
Then, MVC = Lysate Sensitivity(λ)
Endotoxin Limit
= 0.125 EU/mL
0.2EU/mg
MVC = 0.625 mg/mL
 BET Product validation

 The validation for LAL compatibility of a drug product at a test condition

where an Endotoxin standard is detected with the same efficiency in a
test sample as it is in the LRW.

 Product Validation steps:

Phase I: Preliminary Screening is done to determine the

Non - Interfering Dilution (NID)/ Concentration
of the product

Phase II: The goal of a BET validation is to find a valid,

compatible range (Concentration or Dilution) for
routine LAL testing of the material in question.
 Phase I: Preliminary Screening

• Prepare Dilutions / Conc. of product up to MVD/MVC such as

MVD/16, MVD/8, MVD/4, MVD/2, MVD
or
16MVC, 8MVC, 4MVC, 2MVC, MVC
• Let us say MVD is 800 and MVC is 0.625 mg/mL then:

Sample Undil. MVD/16 MVD/8 MVD/4 MVD/2 MVD

Sample
Dilution 1:1 1:50 1:100 1:200 1:400 1:800

Conc. 500 10 5 2.5 mg/mL 1.25 0.625

mg/mL mg/mL mg/mL mg/mL mg/mL
 Preliminary Screening
Dilution Tubes LRW CSE Sample LAL
(L) (L) (L) (L)
Blank NWC 1,2 100 - - 100
Undiluted PPC 3,4 - 10 (20λ) 100 (Undil.) 100

NPC 5, 6 - - 100 (Undil.) 100

MVD/8 PPC 7, 8 - 50 (4λ) 50 (MVD/16) 100
(1:100)
NPC 9. 10 50 - 50 (MVD/16) 100
MVD/4 PPC 11, 12 - 50 (4λ) 50 (MVD/8) 100
(1:200)
NPC 13, 14 50 - 50 (MVD/8) 100
MVD/2 PPC 15, 16 - 50 (4λ) 50 (MVD/4) 100
(1:400)
NPC 17, 18 50 - 50 (MVD/4) 100
MVD PPC 19, 20 - 50 (4λ) 50 (MVD/2) 100
(1:800)
NPC 21, 22 50 - 50 (MVD/2) 100
 Screening for Non Interfering Dilution/
Concentration
Sample Undil. 1:100 1:200 1:400 1:800
Dilutions (MVD/8) (MVD/4) (MVD/2) (MVD)
Sample
Conc. 500 5 2.5 1.25 0.625
(mg/mL)
Unspiked
(NPC)
-- -- -- -- --

Spiked
-- -- ++ ++ ++
(PPC)

• Non Interfering Dilution (NID) : 1: 200

• Non Interfering Conc. (NIC) : 2.5mg/mL
• Test Dilution : 1: 400
 Validation of Product
Endotoxin / Water Endotoxin / Product
0.25 0.125 0.06 0.03 0.25 0.125 0.06 0.03

+ + + - + + - -
+ + - - + + - -
+ + - - + + - -
+ + + - + + - -
GM endpoint = 0.085 EU / mL GM endpoint = 0.125 EU/mL

Successful validation requires that both series confirm label claim

+/- two fold dilution
 Product Validation
Groups Test LRW Sample CSE LAL Results
Sample
NWC NWC 100µL ─ - 100µL ─

2λ 50µL ─ 50µL (4 λ) 100µL ++++

λ 50µL ─ 50µL (2λ) 100µL ++++

CSE/LRW
λ/2 50µL ─ 50µL (λ) 100µL ----

λ/4 50µL ─ 50µL (λ/2) 100µL ----

NPC NPC 50 µL 50 µL (1:200) - 100µL ----

PPC (2λ) ─ 50 µL(1:200) 50µL (4 λ) 100µL ++++

PPC (λ) ─ 50 µL(1:200) 50µL (2λ) 100µL ++++

Product /
CSE PPC (λ/2) ─ 50 µL(1:200) 50µL (λ) 100µL ----

PPC (λ/4) ─ 50 µL(1200) 50µL (λ/2) 100µL ----

 Product is Validated
• If endpoint of CSE in product is within + one two fold of endpoint in water.

• GM end point Concentration = Antilog (∑ e / f)

• GM in water = log(0.125) + log(0.125) + log(0.125) + log(0.125)

4
= Antilog [ (-0.903) + (-0.903) + (-0.903) + (-0.903) ]
4
= Antilog (-0.903)

= 0.125 EU/mL

• GM in product = log(0.125) + log(0.125) + log(0.125) + log(0.125)

4
= Antilog [ (-0.903) + (-0.903) + (-0.903) + (-0.903) ]
4
= Antilog [(-0.903)]

= 0.125 EU/mL
 Product Re-validation

• New Products – 3 Batches

• Change of LAL vendor – 1 Batch validation
• Change in Formulation
 Routine Testing

• 50-50 Method

• Hot Spike Method

 Routine testing
 Negative Water Control (NWC) : LRW + LAL

 Positive Water Control (PWC) : LRW + CSE (2λ) + LAL

 Negative Product Control (NPC) : Sample + LAL

 Positive Product Control (PPC) : Sample + CSE (2λ) + LAL

 50-50 Method
Test Tube LRW CSE (4λ) Sample LAL
No.
NWC [ 1, 2] 100µL - - 100µL

PWC [ 3, 4] 50µL 50µL - 100µL

NPC [ 5, 6] 50µL
- 50µL (MVD/2) 100µL
MVD
PPC [ 7, 8]
- 50µL 50µL (MVD/2) 100µL
MVD
NPC [ 9, 10]
50µL - 50µL (MVD/4) 100µL
MVD/2
PPC [ 11,12]
- 50µL 50µL (MVD/4) 100µL
MVD/2
 Hot Spike Method
Test Tube No. LRW CSE Sample LAL

NWC [ 1, 2] 100µL - - 100µL

PWC [ 3, 4] 100µL 10µL(20λ) - 100µL

NPC [ 5, 6]
- - 100µL (MVD) 100µL
MVD
PPC [ 7, 8]
- 10µL (20 λ) 100µL (MVD) 100µL
MVD
NPC [ 9, 10]
- - 100µL (MVD/2) 100µL
MVD/2
PPC [ 11,12]
- 10µL (20 λ) 100µL (MVD/2) 100µL
MVD/2
 Expected Results
Test Tube PASS INTERFERES FAILS REPEAT
No.

NWC ─ ─ ─ ─ ─ ─
─ ─

PWC + + + + + + + +

NPC ─ ─
─ ─ + + ─ +

PPC ─ ─
+ + + + + +
 Interpretation of Results
Sample Dilution Endotoxin Content

MVD/16 1 : 50 < 0.012 EU/mg

MVD/8 1 : 100 < 0.025 EU/mg
MVD/4 1 : 200 < 0.05 EU/mg
MVD/2 1 : 400 < 0.10 EU/mg
MVD 1 : 800 < 0.20 EU/mg

• Endotoxin Content = Lysate Sensitivity X Dilution factor

Potency
 Applications of LAL test
• Large Volume Parenterals (LVP)
• Multiple ingredients drugs
• Small Volume Parenterals (SVP)
• Radiopharmaceuticals
• Biological
• Water System validation
• Validation of Dry Heat Sterilizers
• Medical Devices
 Contact Us To Know More
Ravinder Singh
(Technical Executive)

Charles River Laboratories India Pvt. Ltd.

307, Shaswat, Ellisbridge,
Ahemdabad – 380 006
Phone : 079-26407713
Mobile : 9417002472
Fax : 079 –26406992
Email : ravinder.singh@crl.com & ahdoffice@crl.com
Thank You