Protocol for Hold Time Study of Prepared Control Standard Endotoxin (CSE) : Pharmaceutical Guidelines

Protocol for Hold Time Study of Prepared Control Standard Endotoxin (CSE)

Learn how to validate the prepared control standard endotoxin (CSE) for hold time period of 21 days.

1.0  Objective

       Objective of this protocol is to provide documented evidence through the scientific data to establish and verify the label claim of CSE after reconstitution and subsequent storage upto 21 days.

2.0  Scope

       The scope of this protocol is to evaluate the hold time of CSE after reconstitution and subsequent storage upto 21 days. This protocol shall also define the reconstitution of CSE, Lysate and effectiveness during storage period as per manufacturers recommendation.

3.0  Reference Document

       Following documents are referred during preparation of the protocol.

4.0  Responsibility

4.1  Quality Control: Preparation,review and execution of protocol.
4.2  Quality Assurance: Review and approval of protocol.

5.0  Procedure

5.1  Pre-Requisite

•  Depyrogenated glassware and accessories.
•  Reference documents.
•  Certificate of ready to use material.
•  Reagents and materials
•  Testing facility and equipments.

5.2  Procedure For Hold Time Study

•  Reconstitute the lysate with LAL Reagent water (LRW) as per the instruction given by the.
•  Reconstitute the CSE with LRW as per the manufacturer’s instruction and vortex it for the specified time period.
•  Dilute the CSE with LRW to get 2l, l, l/2, l/4 EU/ mL concentration of Endotoxin and perform the test as describe below.

CSE Conc.
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
- ve control
100 ml
100 ml

•  Vortex gently each tube and insert in triobloc maintained at a temperature 37°C ± 1°C and incubate for 60 min ± 2 min.

•  After completion of incubation remove the tubes from triobloc and observe the tubes by inverting smoothly at 180° for positive or negative results. 

•  A firm gel clot that maintains its integrity upon inversion at 180° indicates positive results. 
•  Tubes, which indicate no firm gel or show visible increase in turbidity or viscosity, are reported as negative. 
•  Find out the end point, the lowest concentration that gives positive results is the end point. Find out the log end point.
•  Find out the Geometric mean as follows,                                           
Geometric Mean = Antilog --------

e = log end points F = Number of replicates
•  Perform the CSE curve upon reconstitution and there after every 3rd day upto 21 days. If holiday falls on the test date perform the CSE curve on next working day.
•  Storage shall be done at 2 – 8°C upto end of the hold time study.

6.0  Acceptance Criteria: 

Labelled claim is verified and found ok, if Geometric Mean of the endpoint confirms between 0.5λ and 2λ of the label claimed sensitivity of lysate.

7.0  Conclusion

After complete evaluation of the hold time study for CSE a final hold time study summary report shall be prepared which should essentially contain discussion and conclusion which clearly determine the hold time period for CSE.

8.0  Abbreviations

SOP : Standard operating procedure
m l : Micro litre
CSE : Controlled standard endotxin
λ : Lambda
° C : Degree Celsius
LRW : LAL reagent water
EU/mL : Endotoxin unit per milliliter
LAL : Limulus Amebocyte lysate

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.
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