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
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.
6.1 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.
|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.
|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.
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 Standard Operating Procedure
QA Quality Assurance
QC Quality Control
GPT Growth Promotion Test
LAF Laminar Air Flow
°C Degree Celsius
NLT Not Less Than
PPC Positive Product Control
NPC Negative Product Control
LRW LAL Reagent Water
EU Endotoxin Unit
µl Micron litre
λ Sensitivity of the Lysate
8.0 ANNEXUREURE :
|Annexure No.||Title of Annexure||Format No.|
|Annexure – I||Endotoxin Challenge Test Report|
9.0 DISTRIBUTION :
- Master Copy Quality Assurance Department
- Controlled Copy No. 01 Quality Assurance
- Controlled Copy No. 02 Quality Control (Microbiology)