Issues in the Bacterial Endotoxin Test

Click to edit Master title styleIssues in the Bacterial Endotoxin Test

National Institutes for Food and Drug ControlGao Hua

Confidential for restricted use only

Click to edit Master title style

1. Brief introduction of bacterial endotoxin test

2. Contents and relevant requirements in the experiment

3. Analysis in case of abnormalities in the results

4. Issues to be considered in the application

Confidential for restricted use only

Click to edit Master title styleBrief Introduction• Bacterial endotoxin is one of components of the cell

wall of Gram negative bacteria: LPS

• It is released after the death of the cells.


• Properties of bacterial endotoxin:

Heat resistant It is not easily deactivated (180℃, 2 hrs; 250℃, 30 mins)

Molecular polarity It is prone to aggregate and absorb, and should be sufficiently mixed

High pyrogenicity It acts on human body to cause pyrogen reactions, such as fever, chill and nausea, and even death.

Brief Introduction

Click to edit Master title styleRelations between the pyrogen and bacterial endotoxin

• Pyrogen: All substances causing pyrogen reactions are pyrogen.

• Bacterial endotoxin is only one pyrogen. Not every pyrogen has lipopolysaccharide structure, but all known endotoxins have pyrogen activity.

PyrogenBacterial endotoxin

Click to edit Master title styleRelations between the pyrogen and bacterial endotoxin

• The acceptable view point for drug production and quality control under GMP conditions: no endotoxin means no pyrogen.

• Interpretation: under GMP production conditions, bacterial endotoxin is the key point of quality control of pyrogen.

Click to edit Master title styleDefinition of bacterial endotoxin test

• Bacterial endotoxin test is a method to test or quantify the bacterial endotoxin generated in Gram-negative bacteria with TAL/LAL to evaluate whether the bacterial endotoxin in the test sample complies with the requirement.

Confidential for restricted use only 7

Click to edit Master title styleReaction Mechanism

C factor Activated C factor

Endotoxin

Activated B factorB factor

CoagulaseProclotting

enzyme

Coagulagen Coagulated protein(gel)

β- glucan

Activated G factor

(Bypass reaction)

G factor

Divalent ionpH6.0~8.0

TAL/LAL

Click to edit Master title styleHarmonized Endotoxin Test Worldwide (Internationalization)

• Harmonized standard substances

• Harmonized bacterial endotoxin test

Click to edit Master title styleHarmonized International Standard Substance

Use the potency as the unitIn 1980, the transformation from the weight to potency was completed

ng EU

In 1982, the potency unit EU is used in United States Pharmacopeia

Endotoxin Unit (EU)

Click to edit Master title styleHarmonized International Standard Substance

• U.S. Endotoxin Unit (EU)

• WHO International Unit (IU)

• WHO has established the endotoxin international standard substance

The first batch of international standard substance in 1985 0.7I U=1.0 EU

The second batch of international standard substance in 1995 1.0 IU=1.0 EU

The third batch of international standard substance in 2013 1.0 IU=1.0 EU

Click to edit Master title styleHarmonized Endotoxin Test Worldwide (Internationalization)

• Harmonized method: Since January 2001

United States Pharmacopeia (USP)European Pharmacopoeia (EP)Japanese Pharmacopoeia (JP)

Harmonized bacterial endotoxin test (BET) was implemented, i.e., “International Harmonization Plan of Bacterial Endotoxin Test (ICH)”.

• Chinese Pharmacopoeia was in line with the world in 2005• In Chinese Pharmacopoeia of 2010 edition and 2015 edition,

the endotoxin test is also consistent with the harmonization plan.


• Bacterial endotoxin test includes:

• Any method can be used for the test. When there is disputein the result, the result of the gel-clot technique shall prevail.

Kinetic-chromogenic assayEndpoint-chromogenic assay

Classification of bacterial endotoxin test

(Quantitative experiment)

Gel-clottechnique

Semi-quantitative test

Limit test

Turbidimetric technique

Photometric technique

Chromogenic technique

Kinetic-turbidimetric assayEndpoint-turbidimetric assay


Contents and relevant requirements in the

experiment

Click to edit Master title styleInvolved Contents

• Staff• Reagents and consumables• Instruments• Experiment environment

Click to edit Master title styleStaff and Laboratory Qualification

Experiment staff is required to participate in relevant training of bacterial endotoxin to acquire relevant record or certificate

The laboratory is required to participate in external capability test (PTS)

4 times for the first year

Twice for the second year

Once thereafter

Click to edit Master title styleStaff and Laboratory Qualification

• National Institutes for Food and Drug Control has been always participating in the bacterial endotoxin test capability test plan organized by the Charles River Corporation worldwide.

• The results were all“satisfactory”.

• This suggests that the laboratory is capable of accurately determining the bacterial endotoxin


• National Institutes for Food and Drug Control provides laboratory test review of bacterial endotoxin (gel-clot technique and kinetic-turbidimetric technique)

• Domestic laboratory can contact the quality management department to participate, 010-53851487

Click to edit Master title styleReagents and Consumables

• Standard substances

• Tachypleus Amebocyte

Lysate

• Water for test

• Test tubes

• Pipette tip

• Assay plate, etc.


• National standard substance of bacterial endotoxinIt is used to calibrate working standard

to calibrate the sensitivity of TAL/LAL and arbitration for relevant experiments

It can be used for multiple times (Kept in a week in refrigeration and 4 weeks in cryostorage)

• Working standard of bacterial endotoxinCalibrate the potency with national standard endotoxin substance as the standardIt is used for relevant experimentsIt is for one-time use

• The standard substance produced by National Institutes for Food and Drug Control should be used

Reagents and Consumables


TAL/LAL

• Quality report of the manufacturer

• The sensitivity retest should be performed before using gel-clot TAL/LAL

• The standard curve reliability verification must be performed before using the photometry TAL/LAL


Water for test

• It should conform to the standard for sterile water for injection, with the content of endotoxin lower than 0.015 EU/mL (for gel-clot technique) or lower than 0.005 EU/mL (for photometry).

• It has no interference to the endotoxin test.

• It cannot be replaced with other water


Test tubes

• Materials: no absorption to endotoxin

• Rinsing verification: no interference to bacterial endotoxin test


Pipette tip, assay plate• The quality test report of the manufacturer requires

sterile without endotoxin• No absorption, no interference• Box-packed pipette tips: the verification for expiry

period is performed after the opening

Click to edit Master title styleInstrument and Equipment

• Thermostatic water bath• Bacterial endotoxin quantitative analyzer• Thermostatic drying oven• Micropipettor, etc.


Calibration requirements for thermostatic water bath• Temperature calibration: 37℃• Using area:

About 3 cm under water, the whole water surface

• Using time: an hour


Quantitative analyzer (Microplate reader) performance verification contents: • Accuracy of test wavelength: 405 nm and other

wavelength (as per the requirements)• Baseline stability• Reaction consistency• Temperature calibration:

37℃, 1 h


Quantitative analyzer (microplate reader) software verification:

• Reaction mechanism

• Formula formulation

• Accuracy of software calculation, etc.

It is completed by software manufacturer, with official written report provided


Quantitative analyzer (microplate reader) confidential content control: • Windows login password is set up for the computer• Software setup:

Login password for every personPrivilege of everyoneEnable audit trailTo ensure the integrity of the data


Calibration of thermostatic drying oven• Temperature calibration: higher than

250℃• Using area: 3 layers (15 points)• Using time: 0.5 hour

• Perform endotoxin deactivation testUse deactivation verification standard substance: After the drying, the endotoxin activity should at least decrease by 3 Ig values


Micropipettor• Including single-channel,

multichannel, fixed, adjustable, continuous sample loading pipettors

• It should conform to the requirements of the accuracy and precision of EDQM on the pipettor

• Documents: NEW Annex 6Qualification of piston pipettes

Click to edit Master title styleEnvironment Control

Environment requirements• Clean environment, without

endotoxin contamination

• Water bath placementAvoid vibration to the water bathCement platform, with door closer installed


Analysis in case of abnormality in the results

(Take the gel limit test as an example)

Click to edit Master title styleGel Limit Test

Test substance tube

Test substance positive control

Negative control

Positive control

Test substance tube: To assay the endotoxin limit in the test substance Test substance positive control: To verify no inhibitory interference on

the sample, and avoid that the negative result of test sample is false negative result

Negative control: No endotoxin contamination in the system Positive control: Systemic overall conditions conform to the requirements

of endotoxin experiment

The control success is the necessary conditions for the experiment success.That is, the experiment is effective only after the negative control is negative, and the positive control is positive, and the positive control of test substance is positive.

+ + -- + +

Click to edit Master title styleAnalysis in Case of Abnormal Results

When the negative control is “+”

When the positive control is “-”

When the test substance positive control is “-”

Cause analysis: There is endotoxin contamination or interferential factors, etc.

When the test substance is “+”, initiate “suspicious result processing procedure”

• Exclude false positive, etc.


Issues to Be Considered in the Application of Bacterial

Endotoxin Test


Click to edit Master title styleIssues to Be Considered in the Application of Bacterial Endotoxin Test

Caution should be taken in the formulation of limit value

Precautions in the experiment The method to exclude interferential factors Processing method and verification of special test

substance Requirements for the establishment of bacterial

endotoxin test



Thank you！
