1.0 OBJECTIVE
To lay down the procedure for validation of depyrogenation oven using Endotoxin indicator vial.2.0 SCOPE
This procedure is applicable to validate the oven, for depyrogenation cycle.3.0 RESPONSIBILITY
3.1 Doing: Technical Assistant (Microbiologist)/Executive.3.2 Checking: Executive/ Manager
4.0 ACCOUNTABILITY
Head of the Department5.0 PROCEDURE
FREQUENCY : Every Three Month.5.1 Reconstitute Endotoxin indicator vial with LRW as per the labeled claim vortex for 1 min at every 10min. interval up to 30minutes.
5.2 Distribute the quantity of Endotoxin indicator in 10 nos of the Pyrogen free vial under LAF (equivalent to 10,000 EU/vial).
5.2 Label 1-9 vial and keep one vial for positive control wrapping with Para film.
5.4 Keep all above nine vials at different location in the oven as per location chart and operate the cycle as per SOP.
5.5 Keep one vial unexposed as a +ve control.
5.6 After completion of cycle take all the nine vials from the oven and bring it to room temperature.
5.7 Reconstitute all vials in 1ml LRW and vortex it for five minutes.
(One vial ) For PCè Dilute it up to 0.125 and 0.25 EU/ml
(Nine vial) For Sample èAssuming 3 log reduction (i.e. each vial containing 10 EU/ml). Dilute it up to 0.125 EU/ml with LRW, as per dilution scheme is given in annexure-I.
5.7 Do the LAL test of all dilution of 0.125 EU/ml and positive control of 0.125 & 0.25 EU/ml and a negative control with LRW, in duplicate.
5.8 All 9 vial (0.125 EU/ml) must be negative for gel formation and positive control should be positive.
5.9 Calculate as per the annexure-II and record the results in it.
5.10 Interpretation of results:- The +ve control must be positive & the exposed vial must be negative indicating >3 log reduction of Endotoxin.
5.10.1 If the exposed vial shows +ve results proceed to revalidation.
5.11.2 If revalidation of the exposed vials shows +ve results then rectify the oven problem and repeat the cycle.
5.10.2 Acceptance Criteria :- There should be >3 log reduction of Endotoxin.
6.0 ABBREVIATIONS
6.1 0C = Degree Centigrade6.2 EU =Endotoxin units
6.3 ml= Millilitre
6.4 LAL = Limulus Amebocyte Lysate
6.5 LRW = Lal Reagent Water
6.6 PC=Positive Endotoxin Indicator control
RECONSTITUTE ALL 10 VIALS WITH 1ML LRW.
è For +ve control :-
(10,000 EU/ml) ----à 100 EU/ml --à 1 EU/ml ----à 0.25 EU/ml ----à 0.125 EU/ml
1:100 1 :100 1:4 1:2
èFor exposed 9 vials :-
(Assuming 3 log reduction) i.e. EU/ml ---à 1 EU/ml ----à 0.125 EU/ml
1:8 (Last dilution)
ANNEXURE-I
MICROBIOLOGY LABORATORY
Oven Validation for Dehydrogenation
1. Depyrogenation Cycle Temp.:_______________ Time : ________________
2. Position of Endotoxin Sample è as per location chart.
3. Reagent Information.
LAL : _______________ Endotoxin :__________________ LRW :________________
Sensitivity :____________ Expiry Date:__________________ Expiry Date:_____________
Expiry Date : ___________________ Reconstitution:________________
Block Temp.: Temp (In) :________ Temp (Out) :_________
Time (In) :________Time (Out) :__________
Time (In) :________Time (Out) :__________
Sr. No.
|
Location
|
Results of Last dilution
|
Remarks
| |
1
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2
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1
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2
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3
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4
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5
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6
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7
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8
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9
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+ve Control 0.25
0.125
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-ve Control
| ||||
ANNEXURE-II
MICROBIOLOGY LABORATORY
Oven Validation for Depyrogenation
Date:_________________
Calculation :-
X Log reduction = (Log of recovered EU/ml from PC-Log of recovered EU/ml from sample)
è Log recovered EU/ml from PC =Total EU from PC X Sensitivity of Lysate.
è Log recovered EU/ml from sample = Remaining EU from sample X Sensitivity of Lysate.
Antilog (>3 log reduction )
i.e. =
Remarks :-
Analyst :_________________ Checked By :____________________
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