Bacterial Endotoxin (LAL) Test Procedure

d Standard Operating Procedure (SOP) for Bacterial Endotoxin (LAL) Test in microbiology for pharmaceutical and healthcare laboratories and food industry. With our comprehensive Bacterial Endotoxin (LAL) Test Procedure, you may expedite your testing process and ensure consistent, dependable results.

Procedure for Bacterial Endotoxin (LAL) Test

1.0 Purpose

- The objective of this SOP is to lay down the procedure for Bacterial Endotoxin (LAL) Test.

2.0 Scope

- This SOP is applicable to the Procedure for Bacterial Endotoxin (LAL) Test in Microbiology Department.

3.0 Responsibility

- Microbiologist/Officer QC -Microbiology shall be responsible for:

- Bacterial Endotoxin (LAL) Test as per this SOP.

- Executive QC -Microbiology shall be responsible for:

- Ensuring Bacterial Endotoxin (LAL) Test as per this SOP.

- Head Quality Control (QC) shall be responsible for:

- Ensuring the adherence to this SOP.

4.0 Procedure for Bacterial Endotoxin (LAL) Test

- Precautions during performing Bacterial Endotoxin (LAL) Test:

- Do not freeze the Endotoxin solutions (CSE vial and dilutions), store at 2-8°C.

- Do not vortex or shake LAL Reagent. Mix gently without formation of air bubbles.

- Always use Endotoxin-free articles (like tube, tips) for the testing.

- Depyrogenate the accessories required like assay tubes, dilution tubes and vials, to make free from Endotoxin at 250°C for not less than 60 min.

- Put on the heating block at least 15 – 20 min. prior so as to achieve the set temperature (37±1° C).

- Switch OFF the LAF blower during the incubation period of assay tubes.

- Every new batch of Lysate must undergo Lysate sensitivity verification, before use.

- Requirements for performing Bacterial Endotoxin (LAL) Test :

- LAL reagent

- CSE

- LRW

- Vortex Mixer

- Endotoxin free assay tube 10 × 75 mm

- Endotoxin free dilution tube 13 × 100 mm

- Heating Block

- Micropipette

- Endotoxin-free disposable micropipette tips

- Endotoxin-free glass pipettes

- Vial Opener

- Preparation of CSE Solution for Bacterial Endotoxin (LAL) Test:

- Do not use CSE after 14 days of reconstitution or unless longer period mentioned in the supplier’s COA.

- Remove Aluminium seal of the CSE vial without opening the rubber stopper.

- Carefully remove the stopper. Keep the stopper in a clean surface without touching the inner portion of stopper.

- Reconstitute the lyophilized CSE as per COA to obtain particular concentration of endotoxin.

- Immediately after reconstitution, vortex mix intermittently for 20-30 minutes. Vortex mix vigorously for at least 1 minute prior to each use.

- Further dilution of CSE depends on the endotoxin content /ml after reconstitution.

- For Example: If we receive the CSE with the sensitivity of 200 EU/vial against a particular lysate (as per COA of supplier) reconstitute it with 5mL LRW to get 40 EU/ml concentration and serially diluted as follows (when λ is 0.125).

- Dilution of CSE:

1st Dilution : – 0.1 ml standard endotoxin + 3.9 ml LRW= [1 EU / ml] 8 λ (A)

2nd Dilution : – 1 ml (A) + 1 ml LRW= [0.5 EU /ml] 4 λ (B)

3rd Dilution : – 1 ml (B) + 1 ml LRW= [0.25 EU /ml] 2 λ (C)

4th Dilution – 1 ml (C) + 1 ml LRW= [0.125 EU /ml] λ (D)

5th Dilution -1 ml (D) + 1 ml LRW = [0.06 EU /ml] λ/2 (E)

6th Dilution -1 ml (E) + 1 ml LRW = [0.03 EU /ml] λ /4 (F)

Note: Use this dilution up to 4 λ for routine testing and λ /4 for standard curve calibration.

Reconstitution of LAL Reagent:

- Remove Aluminium seal of the Lysate vial without opening the rubber stopper.

- Tap on the rubber stopper to collect the LAL powder in the bottom of the vial.

- Carefully remove the stopper. Keep the stopper in a clean surface without touching the inner portion of the stopper.

- Reconstitute the lyophilized LAL reagent with LRW as per the COA.

- Do not shake, mix gently avoiding formation of air bubbles and keep aside till clear solution is not visible.

Storage of LAL Reagent:

- Lyophilized LAL reagent should be stored at 2-8°C, Reconstitute the Lysate at the time of use & store it at 2-8°C for 24 hours. If Lysate is not to be used within 24 hrs then store it at -10 to -30 °C for NMT 28 days or as per COA. The Lysate can be freezed and thawed only once.

- Record the receipt of CSE, Lysate and LRW as per format no.: APBC/MB/027/F-01; “BET Reagent Inward Register”.

pH Adjustment of Sample:

- The pH of the test dilution should be 6.0 to 8.0.

- pH may be adjusted as required with Endotoxin-free 0.1 N Hydrochloric acid, or Endotoxin-free 0.1 N Sodium Hydroxide or suitable buffer as recommended by the LAL reagent manufacturer.

- Preparation of Endotoxin free 0.1N NAOH [approx.]: Add 1.0 ml of 1.0 N NaOH in 9.0 ml of LAL Reagent water.

- Preparation of Endotoxin free 0.1 N HCL [approx.]: Add 1.0 ml of 1.0 N HCL in 9.0 ml of LAL Reagent water.

Preparation of Sample Dilution:

- The sample dilution is prepared by the MVD formula.

- Prepare the dilution used in the test using LRW.

- If necessary adjust the pH of the test solution within the pH range 6.0 to 8.0.

- The allowable dilution level or maximum valid dilution [MVD] is dependent on the potency [concentration] of the product, the endotoxin limit for the product and the lysate sensitivity.

- When the endotoxin limit in the substance or preparation being examined is specified in terms of weight or units of active drug.

MVD= Endotoxin Limit [EU/mg or EU/ unit] × Potency

Sensitivity of Lysate [EU/ml]

Potency: Concentration in mg or units per ml, of the drug in the solution tested.

- When the endotoxin limit in the sample preparation is specified in terms of volume.

MVD = Endotoxin Limit [EU/ml] × Potency

Sensitivity of Lysate [EU/ml]

e.g. dilution for Ceftriaxone Sodium

MVD = 0.20 EU/mg × 100mg/ml= 160

0.125 EU/ml

Note: Use MVD/4 dilution for routine testing or as per requirement.

Dissolve 100 mg Ceftriaxone Sodium in 1 ml LRW (a)

Take 0.1 ml from (a) add 3.9 ml LRW (b)

e.g. dilution for Sterile Water for Injection /WFI

MVD = 0.25 EU/ml × 1ml/ml = 2

0.125 EU/ml

Use as such water for testing; without dilution in LRW

Testing Methodology of Bacterial Endotoxin (LAL) Test:

- Each specimen shall be tested in duplicate.

- NWC: Take 100μl LRW

- PWC: Take 50 μl LRW and 50 μl CSE of 4λ.

- NPC: Take 50 μl LRW and 50 μl test dilution.

- PPC: Take 50 μl test dilution and 50 μl CSE of 4λ.

- Add the LAL reagents as mentioned below in the table 1.

- Mix gently and incubate the tubes in heating block at 37 °C ±1 for 60 min.

- After incubation remove the tubes gently from the heating block and slowly invert through 180°C and note the observations.

- If a firm gel has formed, record the results as positive.

- A result is negative, if an intact gel is not formed.

- Record the results and other details as per format no. 2 – “Analytical Test Data Sheet for Bacterial Endotoxin (LAL) Test” in case of Sterile raw material, WFI and Finished product.

Table-1: Sequential addition of the solution (LRW, Product, CSE and Lysate)

Tube	LRW (μl)	Product(µl) Test Dilution	CSE	Lysate (µl)	Expected result
NWC	100	–	–	100	–
PWC	50	–	50 µl of 4λ	100	+
NPC	50	50 (MVD/4)	–	100	–
PPC	–	50 (MVD/4)	50 µl of 4λ	100	+

- Record the consumption of CSE, Lysate as per format no 03 – “Consumption Record of Lysate & CSE” and of LRW as per format no 05; “Consumption Record of LRW”.
Lysate Sensitivity Verification:

- Each new batch of Lysate has to be verified for its sensitivity before use.

- Take Lysate vial to check Lysate sensitivity.

- Reconstitute the LAL reagent as directed on the vial with LRW to achieve labeled Lysate sensitivity.

- Prepare CSE dilution of 2λ, λ, λ/2, and λ/4.

- Put sign with date on the CSE vial with date of reconstitution.

- Mark all the reaction Tubes as required and pipette out 100μl of all the 4 CSE dilutions (2λ, λ, λ/2 and λ/4) in quadruplicate.

- In two test tubes, pipette out 100μl of LRW (NWC).

- Add 100μl of reconstituted LAL in all the test tubes.

- Incubate all the tubes at 37 ± 1°C for 60 ± 2 minutes.

- After completion of incubation time, take out reaction tubes from heating block and invert it to 180° in one single smooth motion.

- If a firm gel has formed, record the result as positive.

- If an intact gel is not formed and falls down upon inverting the tube, record the result as negative.

- Test is not valid, unless the lowest concentration of the standard solutions shows a negative result in all replicate tests.

- The test is not valid, If negative control is positive.

- 2 λ should be Positive in all replicate.

- Determine the Geometric mean of end point concentration as follows.

- Geometric mean End point concentration = Antilog (Σe) f

Where (Σe) = Sum of log of end point of all the replicates

f = Number of replicates

- Geometric end point concentration is the measured sensitivity of the LAL reagent in EU/ml

- The end point result should be between 2λ and λ/2.

- Record all the result in format no. 04 -“LAL Reagent Sensitivity Test Record”.

Table-2 : Lysate Sensitivity Verification

CSE Concentration	No. of Replicates
CSE Concentration	1	2	3	4
2λ (0.25)	+	+	+	+
λ (0.125)	+	+	+	+
λ/2(0.06)	+	+	–	–
λ/4(0.03)	–	–	–	–
End point	0.06	0.06	0.125	0.125
Log¹⁰ End point	-1.221	-1.221	-0.903	-0.903
Mean of log¹⁰ end point	-1.062
Geometric mean of log¹⁰endpoint (Antilog of mean log¹⁰ end point)	Lysate Sensitivity= 0.0866EU/ml

REFERENCES : United State Pharmacopoeia; Chapter No. 85.

5.0 Abbreviations :

- MB : Microbiology
- CSE : Control Standard Endotoxin
- °C : Degree Centigrade
- LAL : Limulus Amaebocyte Lysate
- mm : Millimeter
- COA : Certificate of Analysis
- EU : Endotoxin Unit
- ml : Milliliter
- NMT : Not More Than
- Hrs : Hours
- N : Normal
- NaOH : Sodium Hydroxide
- HCl : Hydrochloric Acid
- MVD : Maximum Valid Dilution
- LRW : LAL Reagent Water
- WFI : Water for Injection
- NWC : Negative Water Control
- PWC : Positive Water Control
- NPC : Negative Product Control
- PPC : Positive Product Control
- µl : Microlitre

6.0 Attachments – Bacterial Endotoxin (LAL) Test

Annexure -01 : “BET Reagent Inward Register”

Date of Receipt	Batch /Lot No. of Lysate	No. of Vials Received	Labeled Sensitivity	Expiry Date of Lysate	Batch/Lot No. of CSE	No. of Vials Received	Potency of CSE in EU/ml

Expiry Date of CSE	Quantity of LRW received in ml	Batch/Lot No. of LRW	Expiry Date of LRW	Received By	Checked By

Annexure -02 : “Analytical Test Data Sheet for Bacterial Endotoxin (LAL) Test”

Sample Name:				Batch No.:
Date of Test:				A.R. No.:
Sample : Endotoxin Limit : ____________ EU/mg/ml MVD : ____________ Stock Concentration : ____________ mg/ml MVD/2 : ____________ Sample preparation : ____________ MVD/4 : ____________ 1) Dilute _____ ml of Stock solution with _____ ml of LRW —– (A) 2) Dilute _____ ml of Solution (A) with ________ ml of LRW —– (B) 3) Dilute _____ ml of Solution (B) with ________ ml of LRW —– (C) 4) Dilute _____ ml of Solution (C) with ________ ml of LRW —– (D) to get MVD/4 dilution. Lysate : Lot No.: ____________ Labeled Sensitivity : ____________ EU/ml Expiry : ____________ Confirmed Sensitivity : ____________ EU/ml Reconstituted on: ____________ with _____ ml LRW. Sensitivity tested on: ____________ LRW : Lot No.: ____________ Expiry: ____________ CSE : Lot No.: ____________ Expiry : ____________ RSE / CSE Ratio : ____________ EU Vial Content: ____________ EU Reconstitution volume: ____________ ml Stock concentration ________ EU/ml 1) Dilute ______ ml of Stock solution with ______ ml of LRW (A) 2) Dilute ______ ml of Solution (A) with ______ ml of LRW to get Concentration of 4l Incubation Time ____________ Incubation Temperature ____________
Observation :
Tube No.	Sample	Volume in µl					Results	Remarks
		Sample	LRW		CSE	Lysate
1, 2	NWC					100
3, 4	PWC					100
5, 6	PPC					100
7, 8	SAMPLE (NPC)					100

Conclusion: Sample Complies/Does not Complies for BET as per IP/BP/USP/EP/In-House Specification.

Analyzed By: Checked By:

Date: Date:

Annexure -03 : “Consumption Record of LAL Reagent”

Lysate Quantity

CSE Quantity

Lot No.

Expiry Date

S.N.

Lysate

CSE

Done By

Checked By

Reconstituted on

Date of Opening

Stock Qty.(µl)

Used Qty.(µl)

Balance Qty.(µl)

Reconstituted on

Date of Opening

Stock Qty.(µl)

Used Qty.(µl)

Balance Qty.(µl)

Annexure -04 : “LAL Reagent Sensitivity Test Record”

Name of Reagent

Lot No.

Labeled sensitivity (l)

Confirmed

Sensitivity(l)

Expiry Date

Reconstitution Date

Lysate

CSE

LRW

–

Incubation Temp:

Incubation Time:

CSE :

RSE / CSE Ratio : _______ EU/_______ Vial Content _______ EU

Reconstitution volume: _______ ml

Stock concentration _______ EU/ml ( vortex for 10 min before use)

· _______ml. Stock + ____ml. LRW——A ( ) Vortex for 30 Seconds

· _______ml. A + _______ml. LRW——-B ( ) Vortex for 30 Seconds

· _______ml. B + _______ml. LRW——-C ( ) Vortex for 30 Seconds

· _______ml. C + _______ml. LRW——-D ( ) Vortex for 30 Seconds

· _______ml. D + _______ml. LRW——-E ( ) Vortex for 30 Seconds

Vortex Mixture ID:

Observation :

Replicate No.

CSE Dilutions (EU/ml)

LRW

(Negative Control)

2.0 λ

(___)

End Point

(EU/ml)

— λ

(___)

Log¹⁰ End Point

λ/2

(___)

2.0 λ

(___)

— λ

(___)

λ/2

(___)

λ/4

(___)

Mean

Antilog

Acceptance Criteria: The geometric mean end point concentration must be greater than or equal to 0.5 l or λ/2 and less than or equal to 2l.

Conclusion: Complies / does not Complies with labeled LAL sensitivity.

Analyzed By: Checked By:

Date: Date:

Annexure -05 : “Consumption Record of LRW”

Lysate Quantity
Lot No.
Expiry Date
S. No	Date of Opening	Stock Qty.(µl)	Used Qty.(µl)	Balance Qty.(µl)	Done By	Checked By