sop for endotoxin challenge test

1.0 OBJECTIVE :
To lay down a procedure for Endotoxin Challenge Test.
2.0 SCOPE :
This SOP is applicable for Endotoxin Challenge Test in Microbiology Lab. Of Quality Control
Department
3.0 RESPONSIBILITY :
3.1 Officer / Executive – QC (Microbiology)
4.0 ACCOUNTABILITY :
4.1 Head – QC
5.0 DEFINITION :
The Endotoxin Challenge Vial (ECV) is used in the validation of dry heat depyrogenation cycles. The ability of a particular oven / Tunnel cycle to destroy / inactivate endotoxin is measured by comparing the endotoxin level in baked ECVs vs unbaked control ECVs.
6.0 PROCEDURE:

6.1 material & Instruments :
6.1.1 Limulus Amebocyte Lysate Reagent
6.1.2 Endotoxin Indicator Vial (100000 EU/ml)
6.1.3 LAL Reagent Water
6.1.4 Ampoules or Vial according to requirement
6.1.5 Depyrogenated Dilution Tubes (13×100 mm)
6.1.6 Depyrogenated Assay Tubes (10×75 mm)
6.1.7 Micropipette with Pyrogen free tip (20-200 µl)
6.1.8 Micropipette with Pyrogen free tip (100-1000 µl)
6.1.9 Vortex Mixer
6.1.10 Heating Block
6.2 Preparation Of Challenge Vials :
6.2.1 Reconstitute the Challenge Vial of Endotoxin (100000 EU/ml) with 10 ml LRW to yield 10,000 EU/ml) and vortex according to manufacturer”s instructions.
6.2.2 Transfer 0.1 ml aliquot in to ampoules or vials used for Challenge Test (10000 EU/Vial).
6.2.3 Dry the ampoules or vials in Laminar Air Flow for overnight . Each ampoule or vial now contains 10000 EU/Vial. Mark the above prepared Ampoules/Vials as 1 to 10 numbers.
6.2.4 Keep at least 1 Ampoule/Vial as positive control (do not expose through Oven / Tunnel).
6.2.5 Mark the remaining ampoules/vials as NPC.
6.2.6 Expose these Ampoules/Vial to appropriate location in DHS/Tunnel as per its typical Depyrogenation Cycle.
6.3 Dilution Of Positive Control Ampoule / Vial :
6.3.1 Reconstitute the Ampoules/Vials with 1 ml LRW and vortes vigorously for 15 minutes and each subsequent dilution for 2-4 minutes.
6.3.2 Now the concentration of Endotoxin in the PPC will be 10000 EU/ml.
6.3.3 Prepare 1:20 dilution of the 10000 EU/ml to obtain 500 EU/ml.
6.3.4 Prepare 1:20 dilution of the 500 EU/ml to obtain 25 EU/ml.
6.3.5 Prepare 1:25 dilution of the 25 EU/ml to obtain 01 EU/ml.
6.3.6 From the above 1 EU/ml preparation, prepare a two fold dilution series up to 2λ, λ, λ/2, λ/4, where λ = Labelled Lysate Sensitivity, λ = 0.125 EU/ml.

DILUTION TABLE

Sr. No.	ENDOTOXIN	LRW	ENDOTOXIN CONCENTRATION (EU/ML)
1.	10000 EU/Vial	1 ml	10000 EU/ml
2.	0.1 ml of 10000 EU/ml	1.9 ml	500 EU/ml
3.	0.1 ml of 500 EU/ml	1.9 ml	25 EU/ml
4.	0.1 ml of 25 EU/ml	2.4 ml	01 EU/ml (8 λ)
5.	1 ml of 01 EU/ml	1 ml	0.5 EU ml (4 λ)
6.	1 ml of 0.5 EU/ml	1 ml	0.25 EU ml (2 λ)
7.	1 ml of 0.25 EU/ml	1 ml	0.125 EU ml (λ)
8.	1 ml of 0.125 EU/ml	1 ml	0..0625 EU ml (λ/2)
9.	1 ml of 0.0625 EU/ml	1 ml	0.0325 EU ml (λ/4)

6.4 dilution And Test Of Npc Ampoules / Vial :
6.4.1 It is assumed that three log reduction is achieved after exposure of ampoules / vials in oven /Tunnel, the Endotoxin concentration in the vial is 10 EU/Vial now.
6.4.2 Reconstitute each ampoules/Vials with 1 ml LRW and vortes vigorously for 15 minutes and the Endotoxin concentration in the each ampoule or vial is 10 EU/ml.
6.4.3 Take 0.1 ml from above each vials or ampoules of 10 EU/ml in assay tubes as duplicate and add 0.1 ml Lysate which have the potency 0.125 EU/ml.
Dilution Table

Sr. No.	Endotoxin	Lysate	LRW	Endotoxin Concentration (Eu/ml)
1.	10 EU/Vial or Ampoule Assumed	–	1 ml	10 EU/ml
2.	0.1 ml of 10 EU/Vial	0.1 ml	–	01 EU/ml

6.5 LAL Test Procedure :
6.5.1 Test the two fold dilution series prepared from the positive controls ampoules / vial in duplicate.
6.5.2 Test the 10 EU/ml dilutions prepared from each of exposed ampoules / Vial.
6.5.3 Test should be carried out in Clean depyrogenated 10×75 mm assay tubes only.
6.5.4 Protocol for Positive Control :

Sr. No.	Dilutions	CSE Dilution Used	LRW	Lysate in µl	No. of Replicates
1.	2λ	100 µl of 2λ	–	100 µl	2
2.	λ	100 µl of λ	–	100 µl	2
3.	λ/2	100 µl of λ/2	–	100 µl	2
4.	λ/4	100 µl of λ/4	–	100 µl	2
5.	Negative Water Control (NWC)	–	100 µl	100 µl	2

6.5.5 Protocol for Negative Product Control (Challenged Ampoules/Vial):

Ampoule No.	Dilutions	Endotoxin Indicator Dilution (01 EU/ml)	Lysate in µl	No. of Replicates
1.	NPC	100 µl	100 µl	2
2.	NPC	100 µl	100 µl	2
3.	NPC	100 µl	100 µl	2
4.	NPC	100 µl	100 µl	2
5.	NPC	100 µl	100 µl	2
6.	NPC	100 µl	100 µl	2
7.	NPC	100 µl	100 µl	2
8.	NPC	100 µl	100 µl	2
9.	NPC	100 µl	100 µl	2
10.	NPC	100 µl	100 µl	2
11.	NPC	100 µl	100 µl	2
12.	NPC	100 µl	100 µl	2

6.6 Calculation :
6.6.1 Formula :
Endotoxin Concentration = Lysate sensitivity × Reconstitued Volume × Dilution
For Unbaked (Positive Control) Vials = 0.125 EU/ml × 1 ml/Vial × 10,000 = 1250 EU/ml
For Baked (Heat Treated) Vials = 0.125 EU/ml × 1 ml/Vial × 1 = 0.125 EU/ml
Calculate the minimum log reduction as follows :
Minimum Log Reduction = Log Endotoxin Concentration of the Unbaked Control – Log Endotoxin Concentration of the Baked Vials.
OR
Minimum Log Reduction = Log value of recovered Endotoxin from positive control – Log value of recovered sample from heat treated sample.
6.7 Interpretation Of Results / Acceptance Criteria :
6.7.1 Test results are valid of recovery of Endotoxin in non exposed vials is with in a two fold dilution of the labeled claim.
6.7.2 The depyrogenation cycle is considered as successfully validated if there is more than 3 log reduction is achieved in challenge Endotoxin vials exposed in to Oven/ Tunnel at specified place. Heat Exposed Vials.
6.7.3 For a valid Depyrogenation cycle, the PPC must be positive and NPC’s must be negative indicating a greater than 3 log reduction of pyroburden.
6.7.4 Record the “ Endotoxin Challenge Test Record in Annexure –I.
7.0 ABBREVIATION :

sop for endotoxin challenge test

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