Sop for Method validation report for bacterial endotoxin test

PRODUCT NAME
BATCH NUMBER
DATE OF TESTING
DATE OF COMPLETION

REPORT CONTENTS

S. No.	Title	Page No.
1.0	Pre-Approval
2.0	Objective
3.0	Scope
4.0	Responsibility
5.0	Training record
6.0	Observation
7.0	Acceptance criteria
8.0	References
9.0	Documents to be attached
10.0	Deviation from pre-defined specification, if any
11.0	Change control, if any
12.0	Review (inclusive of follow up action, if any )
13.0	Conclusion
14.0	Recommendation
15.0	Abbreviations
16.0	Revision history
17.0	Report post -approval

1.0 REPORT PRE -APPROVAL:
PREPARED BY:

DESIGNATION

NAME

SIGNATURE

DATE

OFFICER/EXECUTIVE

(MICROBIOLOGY)

REVIEWED BY:

DESIGNATION	NAME	SIGNATURE	DATE
EXECUTIVE/MANAGER (MICROBIOLOGY)
HEAD (PRODUCTION)
HEAD (QUALITY CONTROL)

APPROVED BY:

DESIGNATION

NAME

SIGNATURE

DATE

HEAD

(QUALITY ASSURANCE)

2.0 OBJECTIVE:
To establish a documented evidence and a procedure which will enable to show that the product should not giving any inhibitory properties of Bacterial Endotoxin validation
3.0 SCOPE:
Purpose of this validation activity is to establish documentary evidence that the performance of the test method used for Bacterial Endotoxin testing of a product by Gel clot method, is consistent and reproducible without showing any interference. This validation activity is limited to the validation of Bacterial Endotoxin testing of a product by using Gel clot method.

4.0 RESPONSIBILITY:
The Validation Group, comprising of a representative from each of the following departments, shall be responsible for the overall compliance of this report.

DEPARTMENTS	RESPONSIBILITIES
Quality Control	· Preparation, Review of the method validation Report for Bacterial Endotoxin test. · Protocol Training. · Sampling as per protocol. · To conduct the validation study as per protocol. · To perform the microbiological analysis.
Quality Assurance	· Co-ordination with Quality Control, Production and Engineering to method validation Report for Bacterial Endotoxin test.
Production	· Review & Approval of Protocol. · To co-ordinate and support method validation Report for Bacterial Endotoxin test.
Engineering	· Co-ordination, Execution and technical support in method validation Report for Bacterial Endotoxin test.

5.0 TRAINING ATTENDANCE RECORD:

S. No.	Name of Trainee	Department Name	Designation	Remark

Inference: ………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

Trainer: 

Sign & Date ………………..

6.0 OBSERVATION
6.1 INITIAL QUALIFICATION (PREPARATORY TESTING)

Reagent detail	LAL	CSE	LRW
Lot No.
Expiry date
Date of Reconstitution/opening
Use before
sensitivity(ʎ)/potency
Make

CSE dilution series:

Tube no.	Volume of CSE added	Volume of LRW added	final concentration

Observation and results:

Heating block ID.
Time of incubation					Time of incubation
Temperature at incubation					Temperature at observation
Replicate no.	Concentration of CSE (EU/ml)				End point	Log₁₀End point	Negative control
	2ʎ	ʎ	ʎ/2	ʎ/4
1
2
3
4
Mean of Log₁₀End point
Geometric mean= Antilog₁₀of mean end point

Legends:

‘+ve’ Gel present	‘-ve’ No gel	ʎ: sensitivity of lysate
LRW: LAL Reagent Water	LAL: Limulus Amoebocyte Lysate	EU: Endotoxin Units

Remark: Geometric mean end point concentration is within / not within the ± two fold of the labeled claim sensitivity of the lysate.

Analyst Checked by
(Sign & Date): (Sign & Date):
6.2 DETERMINATION OF NON-INTERFERING DILUTIONS(NID)

Product Name		Batch No.
Date of testing		Date of observation
Endotoxin limit		Potency of product
Start time of incubation		End time of incubation
Temperature at incubation		Temperature at observation
Heating block ID.

Reagent detail	LAL	CSE	LRW
Lot No.
Expiry date
Date of Reconstitution/opening
Use before
sensitivity(ʎ)/potency
Make

MVD Calculation:
Endotoxin Limit (EU/mg or EU/ml) x Potency
MVD = ————————————————————
Sensitivity of the lysate (EU/ml)

MVD/16 ……………. MVD/8 ……………… MVD/4………… MVD/2………………

CSE Dilution:

Product Dilution:

Tube No.	LRW added	Product solution added	Final concentration of the product

Observation and Results:

Dilution	LRW	Endotoxin	Product	LAL	Result
Dilution	LRW	Endotoxin	Product	LAL	I	II
I (MVD/8 +2 ʎ) PPC	NA	50µl of 4 ʎ	50µl of MVD/16	100µl
I( MVD/8) NPC	50µl	NA	50µl of MVD/16	100µl
II(MVD/4+2 ʎ) PPC	NA	50µl of 4 ʎ	50µl of MVD/8	100µl
II(MVD/4) NPC	50µl	NA	50µl of MVD/8	100µl
III(MVD/2+2 ʎ) PPC	NA	50µl of 4 ʎ	50µl of MVD/4	100µl
III(MVD/2) NPC	50µl	NA	50µl of MVD/4	100µl
IV(MVD+2 ʎ) PPC	NA	50µl of 4 ʎ	50µl of MVD/2	100µl
IV(MVD) NPC	50µl	NA	50µl of MVD/2	100µl
Negative control	100µl	NA	NA	100µl

Conclusion: According to the above results the NID of the product found _________ MVD. As per the safety factor, the
Inhibition and enhancement shall be tested at ________ MVD.

6.3 TEST FOR INTERFERING FACTORS

Start Time of incubation		End Time of incubation
Temperature at incubation		Temperature at observation

CSE Dilution:

Tube No.	Volume of CSE	Volume of LRW	Final CSE concentration
1
2
3
4

Product solution:

Tube No.	Volume of product	Volume of LRW	Final concentration of product
1

Observation and Results:

7.0 ACCEPTANCE CRITERIA:
• The geometric mean end point concentration is the measured sensitivity of the LAL reagent (in EU/ml). if this is not less than 0.5λ and not more then 2λ(±two fold), the labeled sensitivity is confirmed and is used in test performed with this lysate.
• The Non-interfering dilution (NID) is the first set of dilution (PPC) containing 2ʎ Endotoxin that shows a +ve gel.
• There is no observable test interference if the calculated geometric means for both the Endotoxin/water series and Endotoxin / product series confirm lambda (i.e. both GMs are within ± one twofold dilution of the labeled sensitivity).
• The preparation being examined does not comply with the test if positive results are found in both test replicates.
• The preparation being examined complies with the test if negative results are found for both test replicates.

8.0 REFERENCES:
• U.S. Department of Health and Human Services, Public Health Service, Food and Drug Administration, 1987, Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products and Medical Devices.
• Bacterial Endotoxin, V.2.1.9, Bacterial Endotoxin, European Pharmacopoeia, 1987.

9.0 DOCUMENTS TO BE ATTACHED:

• Raw data generated during testing.
• Any Other Relevant Documents.

10.0 DEVIATION FROM PRE-DEFINED SPECIFICATION, IF ANY:
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………

11.0 CHANGE CONTROL, IF ANY:

……………………………………………………………………………………………………………………………………………………………………………………………………………………………………
12.0 REVIEW (INCLUSIVE OF FOLLOW UP ACTION, IF ANY ):

…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

13.0 CONCLUSION :

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

14.0 RECOMMENDATION :

15.0 ABBREVIATIONS:

QC Quality Control
Ltd. Limited
GVP General Validation Protocol
GVR General Validation Report
°C Degree Celsius
QA Quality Assurance
ID. No. Identification Number
NA Not Applicable
MVD Maximum Valid Dilution
NID Non inhibitory Dilution

16.0 REVISION HISTORY:

Revision No.	Change Control No.	Details of Changes	Reason of Changes	Effective Date	Done By
00	Not Applicable	Not Applicable	Initial Document