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 / Executive3.2 Checking: Executive /Manager
4.0 ACCOUNTABILITY
Head of the department.5.0 PROCEDURE
5.1 Material requireda. 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. Micropipettes 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 glassware to be used for the 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 the certificate. Use up to 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 weeks /up to time mentioned on the 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 times the labeled sensitivity (4 λ).
Vortex after each dilution for 1 minute on vortex mixture, use up to 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 whatever 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 whatever is required.
5.3.4 Use Pyrogen-free water for reconstitution of (CSE) Lysate, and dilution of the sample.
5.3.6 Label the 10x75 mm tubes appropriately for positive control, Product positive control, Negative control and Sample.
5.3.7 Perform the test in duplicate.
5.4 Procedure:
5.4.1 With help of micropipette and Pyrogen free Tips, add 0.05ml of Sample (MVD/2 or 2 MVC, whatever 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 the 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 tube, 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 upside 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 are 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 sensitivity5.6.1 Frequency: Every new Lot.
5.6.2 Prepared an 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 the tube for –ve control.
5.6.5 Add 0.1ml of each CS Endotoxin dilution in each tube, 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 a 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 upside down (180° angle ) and note down the results.
5.6.10 Find out the Endpoint of each Endotoxin dilution, means on which sensitivity the test show last +ve results.
5.6.11 Calculate the Geometric mean by the 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 DilutionMVC = 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
Exp. Date:
Labeled Sensitivity:
Reconstitution Date
|
CSE
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:_________________________
|
| 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 :
| ||||||
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