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

1.0 Objective

The 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 up to 21 days.

2.0 Scope

The scope of this protocol is to evaluate the hold time of CSE after reconstitution and subsequent storage up to 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 the preparation of the protocol.

4.0 Responsibility

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

5.0 Procedure

5.1 Pre-Requisite

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

5.2 Procedure For Hold Time Study

• Reconstitute the lysate with LAL Reagent water (LRW) as per the instruction is 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.

Description	Volume	CSE Conc.	LRW	Lysate
2λ	100 ml	2λ	--	100 ml
λ	100 ml	λ	--	100 ml
λ/2	100 ml	λ/2	--	100 ml
λ/4	100 ml	λ/4	--	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 a visible increase in turbidity or viscosity, are reported as negative.
• Find out the endpoint, the lowest concentration that gives positive results is the endpoint. Find out the log endpoint.
• Find out the Geometric mean as follows,
                                                  Ʃe
Geometric Mean = Antilog --------
                                                  F
e = log endpoints F = Number of replicates
• Perform the CSE curve upon reconstitution and thereafter every 3rd day up to 21 days. If a holiday falls on the test date perform the CSE curve on next working day.
• Storage shall be done at 2 – 8°C up to the end of the hold time study.

6.0 Acceptance Criteria 

The labeled 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 a 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
ml: Microliter
CSE: Controlled standard endotoxin
λ: Lambda
° C: Degree Celsius
LRW: LAL reagent water
EU/mL: Endotoxin unit per milliliter
LAL: Limulus Amebocyte lysate

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