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SOP for Lal Test by Gel Clot Method


Standard operating procedure of LAL test by Gel-Clot Method using Limulus Amebocyte Lysate, Pyrogen free water and Control Standard Endotoxin.

1.0  OBJECTIVE

       To define the procedure of LAL test by Gel-Clot Method for  better and error free use of the same.

2.0  SCOPE

       This procedure is applicable to perform LAL test by Gel-Clot method.

3.0  RESPONSIBILITY 

3.1  Doing       : Technical Assistant / Executive
3.2  Checking  : Executive /Manager   

4.0  ACCOUNTABILITY 

       Head of the department.

5.0  PROCEDURE

5.1  Material required

a. Limulus Amebocyte Lysate (0.125 or 0.06 EU/ml)
b. Controlled std. Endotoxin.
c. Pyrogen free water.
d. Vortex mixture.
e. Heating block.
f. Micro pipettes and pyrogen free tips
g. Oven.
h. pH meter.
i. Thermometer.
j. Volumetric flask, Conical flask, pipette, big test tube, 10 X 75mm tubes.
k. Refrigerator.

5.2  Procedure for Depyrogenation

5.2.1  Depyrogenate all the glasswares to be used for test as per SOP.
5.2.2  Reconstitution of Control Standard Endotoxins
5.2.3  Reconstitute the CS Endotoxins with Pyrogen free water as given in certificate. Use upto 4 weeks.
5.2.4  After reconstitution, homogenize the suspension of the  vial of Endotoxins for at least 5 minutes on vortex mixture and record in annexure-III. This reconstituted solution to be stored at recommended storage condition and to be used within 4 week /up to time mentioned on label. Then it should be discarded.
5.2.5  Prepare Endotoxins dilution equivalent labeled sensitivity (λ), One half the labeled Sensitivity (λ/2), twice the labeled sensitivity (2 λ) and four time the labeled sensitivity (4 λ). 
Vortex after each dilution for 1 minute on vortex mixture, use upto 1 week than prepared freshly from the stock solution.

5.3  Product dilution:

5.3.1 
Calculate the MVD for liquid product and MVC for Raw material.

               Limit of Endotoxins X Potency of Product
MVD = ----------------------------------------------------
                  Sensitivity of Lysate ( 0.125 EU/ml)
5.3.2
Find the dilution ratio, MVD/2 or MVD/4 or MVD/8 what ever is required.

                 Sensitivity of Lysate ( 0.125 EU/ml)
MVC = -----------------------------------------------
                          Limit of Endotoxins

5.3.3  Find the dilution ratio, 2 MVC or 4 MVC what ever is required.
5.3.4  Use Pyrogen free water for reconstitution of (CSE) Lysate, and dilution of sample.
5.3.6  Label the 10x75 mm tubes appropriately for positive control, Product positive control, Negative control and Sample.
5.3.7  Perform test in duplicate.

5.4  Procedure:

5.4.1  With help of micro pipette and Pyrogen free Tips, add 0.05ml of Sample (MVD/2  or 2 MVC, what ever is required ) in Sample tubes and PPC (Product positive control ) tubes.
5.4.2  Add 0.05ml of Pyrogen free water to the sample tube and positive control tube.
5.4.3  Add 0.05 ml of CS Endotoxins having a concentration of 2 λ to PPC tube and positive control tube.
5.4.4  Vortex the Endotoxins dilution for 1 minute before adding to the tubes.
5.4.5  Bring the Lysate reagent form the refrigerator and reconstitute it with Pyrogen free water as per label instruction
5.4.6  For reconstitution swirl the Lysate after adding Pyrogen free water gently for 1-2 minutes between two palms of hand. Taking care the foam is not produced.
5.4.7  -ve control i.e. 0.1ml LRW  + 0.1ml lysate
5.4.8  Add 0.1 ml of the Lysate to each tubes, shake the tube for few second and keep the tubes in Heating Block, which is previously kept ‘ON’ and maintained at 36°C to 38°C
5.4.9  Incubate for 60 -62 minute, keep it undisturbed.
5.4.10  After one hour observe, the tube by slowly inverting it up side down (180°angle) 
5.4.11  If firm gel observed : the result is positive
5.4.12  If semi gel or No gel formation observed : the results is negative.
5.4.13  Same way check all the tubes like Negative control tube should show –ve results, Positive control tube and product positive control (PPC)  tube should show +ve results. 

5.5  Interpretations of Results:

Sr No.
-ve Control
+ve Control

Sample

PPC

Interpretation

1
2
1
2
1
2
1
2

1

-ve

-ve

+ve

+ve

-ve

-ve

+ve

+ve

Passes the test

2

-ve

-ve

+ve

+ve

-ve

+ve

+ve

+ve

Repeat the test

3

-ve

-ve

+ve

+ve

+ve

+ve

+ve

+ve

Fail the test

4

-ve

-ve

+ve

+ve

-ve

-ve

-ve

-ve

Repeat the test with using 4 λ of CSE

5

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

Repeat the test with other Pyrogen free water

6

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

Repeat the test with using freshly prepared CSE

5.5.1  Record the results in Annexure – I ( For Insulin Finished Product, Record the results in insulin finished product data sheet.)

5.6  Confirmation of labeled LAL Reagent sensitivity

5.6.1  Frequency : Every new Lot.
5.6.2  Prepared a Endotoxin dilution equivalent to labeled sensitivity (λ), One half of the Labeled sensitivity (λ/2), One fourth of labeled sensitivity (λ/4) and twice the labeled sensitivity (2 λ).
5.6.3  Take four 10x75mm tubes of each sensitivity and label it like 2 λ, λ, λ/2 and λ/4.
5.6.4  Add 0.1ml of Pyrogen free water in tube for  –ve control.
5.6.5  Add 0.1ml of each CS Endotoxin dilution in each tubes, except –ve control tubes.
5.6.6  Add 0.1 ml of  LAL Reagent (Lysate) in each tube.
5.6.7  Vortex all the tubes for few seconds and keep the tubes in heating block which is previously kept ‘ON’ and maintained at 36°C – 38°C.
5.6.8  Incubate for 60-62 minute. Keep it undisturbed.
5.6.9  After one hour, Observe  the tubes by slowly inverting it up side down (180° angle ) and note down the results.
5.6.10  Find out the End point of each Endotoxin dilution, means on which sensitivity the test show last +ve results.
5.6.11  Calculate the Geometric mean by following formula.
                                 Σ e                          
Geometric mean = --------
                                 # ƒ
Where Σe = Sum of Log 10 End Point
          # ƒ  = No. of replicate.
5.6.12  Record the Results in Annexure – II.

5.6.13  Acceptance Criteria:  

The Geometric mean is ± one, two  -  fold dilution of labeled sensitivity. i.e. If labeled sensitivity is 0.125, the Geometric mean is within 0.06 to 0.25

6.0  ABBREVIATION 

MVD = Maximum Valid Dilution
MVC = Maximum Valid Concentration
CSE  =  Control Standard Endotoxins
PPC  = Product positive control 
SOP = Standard operating procedure


ANNEXURE - I
QUALITY CONTROL DEPT.
Page No. : 1 of 2
Product Name:__________________
Batch No.        :__________________
Date of Testing     :__________________
Instrument ID No.:__________________
Product Information
Potency of Product:
Endotoxins Limit :
MVD / MVC
Test Dilution

Reagent Information             
Manufacturer : ________________
Lysate
Lot No.:
Exp. Date:
Labeled Sensitivity:
Reconstitution Date
CSE
Lot No.:
Exp. Date:
Reconstitution Date
LRW 
Lot No      :
Exp. Date :

Incubation : 60 - 62 minutes at 36°C to 38°C Temp.









Analyst                                         
Date :                                                        








Checked by
Date :




ANNEXURE - I

QUALITY CONTROL DEPT.

Page No. : 2 of 2
Product Name:_____________
Batch No.       :_____________
Date of Testing     :__________________
Instrument ID No.:__________________
Observation
Preparation Test Results

1.  Negative control (LRW)
     LRW (0.1 ml) + Lysate (0.1 ml)

2.  Positive LRW control (λ)
     LRW + Std. Endotoxins + Lysate
      (0.05ml + 0.05ml          +  0.1ml)
3.  Test Sample        (1st Tray / lot)
     Test dilution   +   LRW   +   Lysate
            0.05 ml        0.05 ml       0.1 ml

4.  Test Sample         (2nd  Tray / lot)
     Test dilution   +   LRW   +   Lysate
            0.05 ml        0.05 ml       0.1 ml

5.  Test Sample        (3rd  Tray / lot)
     Test dilution   +   LRW   +   Lysate
            0.05 ml        0.05 ml       0.1 ml

6.  Product Positive control (2 λ)
     Test Diln.  +  Std. Endotoxins  +  Lysate
        0.05 ml          0.05 ml of 2 λ       0.1 ml

1.
2.

1.
2.

1.
2.


1.
2.


1.
2.


1.
2.



Where : + ve è Firm gel formation,    - ve è No gel formation
Start Time
Temperature
: ___________
: ___________
Completion Time
Temperature
: ____________
: ____________
The product contains : _______________ EU /                   (Limit :                           ) 
Remarks : Complies / Does not comply


Analyst                                                                                      Checked by
Date :                                                                                         Date :

ANNEXURE - II

QUALITY CONTROL DEPT.

                                            Conformation of labeled LAL Reagent sensitivity

Lysate Mfger :_________________________

Lot No.  :_________________________
Exp.Date:_________________________
Date of Testing      :_______________
Reconstitution Date:_______________
Labeled Sensitivity :_______________

Control Standard Endotoxins

Mfger     :_________________________
Lot No.  :_________________________
Exp.Date:_________________________

Reconstitution Date:_______________
RSE / CSE Ratio    :________________
Potency                  :________________
Vial Content           :________________  

LAL Reagent Water

Mfger     :_________________________


Lot No.  :_________________________
Exp.Date:_________________________


  Observation: Control Standard Curve
Replicate
# ƒ
Endotoxins Concentration

End Point
Log10 End Point
( e )
2 λ
λ
λ / 2
λ / 4
1
2






3






4






-ve Control






Where –ve = No firm gel formation, +ve = Firm gel formation
Start Time:___________, Temp.:__________ Completion Time:___________Temp.:________
Geometric Mean (G.M.) = Antilog Σ e / #ƒ

Result : Conformed Labeled claimed Sensitivity  =                                    
Acceptance Criteria : Labeled claimed Sensitivity of the Reagent is ±  one, two-fold dilution
                                   i.e. If the label sensitivity is 0.125, GM Value is within 0.06 to 0.25
Remark:  Complies / Does not Comply


Analyst:                                                                                   Checked By :
Date    :                                                                                    Date             :

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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