to identify critical method parameters (CMPs), understand their impact on method performance, and facilitate robust, reproducible, and scientifically justified method design in line with ICH Q8, Q9, and Q14 guidelines.

2. Scope

This SOP applies to all method development activities involving chromatographic, spectroscopic, and physicochemical analytical techniques within the Analytical Method Development (AMD) department where DoE is employed to optimize and characterize analytical methods.

3. Responsibilities

• Analytical Scientist: Designs and executes the DoE, interprets results, and identifies MODR.
• Statistician/Design Expert: Provides guidance on model selection, software usage, and statistical analysis.
• Team Lead: Reviews DoE strategy, study results, and performance metrics.
• QA: Verifies compliance with documentation and regulatory standards.
• Head – AMD: Approves DoE-based development conclusions and MODR definition.

4. Accountability

The Head of AMD is accountable for ensuring DoE principles are correctly applied and documented during analytical method development, enabling robust and compliant method lifecycle management.

5. Procedure

5.1 DoE Planning and Strategy

1. Define the objective of DoE (e.g., optimization, screening, robustness).
2. Select appropriate design:
   • Screening: Plackett-Burman, Full Factorial
   • Optimization: Central Composite Design (CCD), Box-Behnken
   • Robustness: Fractional Factorial, Taguchi
3. Document the design plan using Annexure-1: DoE Design Summary.

5.2 Selection of Factors and Levels

1. Identify method parameters to be studied (e.g., pH, flow rate, column temperature, mobile phase composition).
2. Define levels (minimum, center, maximum) based on historical data or feasibility trials.
3. Record all inputs in Annexure-2: DoE Factor Table.

5.3 Execution of Experimental Runs

1. Perform experimental runs as per DoE matrix using validated instruments.
2. Ensure sample prep, injection volume, and detection are consistent across runs.
3. Monitor key responses (e.g., retention time, resolution, tailing, %RSD).
4. Capture output in Annexure-3: DoE Experimental Log.

5.4 Statistical Analysis and Model Interpretation

1. Analyze data using statistical software (e.g., Design-Expert, Minitab, JMP).
2. Generate response surface plots, interaction plots, and contour plots.
3. Determine significance using ANOVA (p-value < 0.05), model R², lack of fit.
4. Identify CMPs and estimate optimal region (MODR).
5. Summarize findings in Annexure-4: DoE Statistical Summary.

5.5 Verification of Optimal Conditions

1. Confirm method performance at optimal DoE-predicted settings.
2. Conduct replicate runs and compare with predicted values.
3. If within acceptable error range, lock the method parameters.
4. Report verification results in Annexure-5: DoE Verification Record.

5.6 Documentation and Approval

1. Compile all DoE-related annexures and include in Method Development Report.
2. Obtain QA and HOD approval before proceeding to validation.
3. Store raw data and software files in designated servers with version control.

6. Abbreviations

• DoE: Design of Experiments
• MODR: Method Operable Design Region
• CMP: Critical Method Parameter
• RSD: Relative Standard Deviation
• Rs: Resolution
• QA: Quality Assurance

7. Documents

1. DoE Design Summary – Annexure-1
2. DoE Factor Table – Annexure-2
3. DoE Experimental Log – Annexure-3
4. DoE Statistical Summary – Annexure-4
5. DoE Verification Record – Annexure-5

8. References

• ICH Q8(R2) – Pharmaceutical Development
• ICH Q9 – Quality Risk Management
• ICH Q14 – Analytical Procedure Development
• USP General Chapter <1032> – Design and Analysis of Biological Assays

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: DoE Design Summary

DoE Type	Objective	Software Used	Date
Central Composite	Optimization of assay method	Design-Expert v13	12/05/2025

Annexure-2: DoE Factor Table

Factor	Low	Center	High	Unit
Flow Rate	0.8	1.0	1.2	mL/min
pH	2.5	3.0	3.5	pH Units

Annexure-3: DoE Experimental Log

Run	Flow Rate	pH	RT (min)	Rs	Tailing
1	1.0	3.0	7.8	2.1	1.1

Annexure-4: DoE Statistical Summary

Response	R²	Adj R²	p-value	Lack of Fit
Rs	0.964	0.951	<0.0001	Not Significant

Annexure-5: DoE Verification Record

Parameter	Predicted	Observed	Deviation (%)	Status
Rs	2.0	1.95	2.5%	Accepted

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added statistical annexures and verification log	ICH Q14 alignment