Automated Method Development Using Analytical Quality-by-Design for Stability Indicating Methods

Significance of the Topic

The stability of pharmaceutical active ingredients under various stress conditions is essential for ensuring product safety, efficacy, and shelf life. Automated, knowledge-driven approaches streamline method development, reduce manual effort, and enhance robustness when monitoring degradation products and impurities.

Aims and Overview of the Study

This work applied an Analytical Quality-by-Design (AQbD) framework using Fusion QbD software in conjunction with Empower 3 to develop a stability-indicating UHPLC method for Tenofovir alafenamide fumarate (TAF). The study aimed to integrate multiple forced degradation samples into a single automated design, define a robust Method Operable Design Region (MODR), and verify method performance under worst-case conditions.

Methodology and Instrumentation

The study comprised four forced degradation preparations of TAF: acid, base, oxidation, and control. Each sample was subjected to specific stress conditions (acid and base at room temperature; oxidation and control at 60 °C) for 3 hours. A two-phase AQbD workflow was implemented:

• Phase 1: Rapid screening of mobile phase pH, column chemistry, and gradient time across all degradation samples to identify promising conditions.
• Phase 2: Focused method optimization targeting column temperature and gradient time to define the acceptable performance region (APR) based on system suitability criteria (peak count, USP resolution, retention factor, tailing).

Used Instrumentation

Key instrumentation included:

• ACQUITY Arc UHPLC system with Quaternary Solvent Manager, Sample Manager, Column Manager
• ACQUITY Photodiode Array Detector (UV detection at 260 nm)
• Columns tested: XBridge Premier BEH Shield RP18; XBridge Premier BEH C18; XSelect Premier CSH Phenyl-Hexyl; XSelect Premier CSH C18 XP
• Empower 3 Chromatography Software and Fusion QbD Method Development Software

Main Results and Discussion

Rapid screening identified the oxidized sample as the worst-case scenario for degradation peak count. Statistical modeling in Fusion QbD predicted optimal pH, column type, and gradient time. Subsequent optimization refined column temperature (41.5–43.5 °C) and gradient length (7–9 min) to meet all system suitability goals. The APR was centered at 43 °C and 8.5 min gradient, yielding robust separation of TAF and its degradants in all stress conditions.

Benefits and Practical Applications of the Method

• Automated integration of multiple degradation samples accelerates method development.
• AQbD approach ensures a scientifically justified operating range, enhancing method robustness and regulatory compliance.
• Improved efficiency reduces time and resources required for stability indicating method validation.

Future Trends and Opportunities

The integration of AQbD workflows with advanced data analytics and machine learning can further automate decision making. Expanding automated forced degradation capabilities to other compound classes and detection techniques (e.g., HRMS) may broaden applicability. Continuous real-time monitoring and adaptive method control represent emerging directions for pharmaceutical quality assurance.

Conclusion

An automated AQbD-driven workflow successfully produced a robust stability-indicating UHPLC method for Tenofovir alafenamide fumarate. The defined MODR and APR provide confidence in method performance across routine and stress conditions, demonstrating the value of automated, knowledge-based method development in pharmaceutical analysis.

Reference

1. Alkhateeb FL, Rainville PD. Analytical Quality by Design Based Method Development for the Analysis of Formoterol, Budesonide, and Related Compounds Using UHPLC-MS. Waters Application Note, 2019.
2. FDA Guidance for Industry: Drug Stability Guidelines (21 CFR 211). 2021.
3. ICH Q1A(R2): Stability Testing of New Drug Substances and Products. International Conference on Harmonization; 2003.
4. Golla VM, Kurmi M, Shaik K, Singh S. Characterization of Degradation Products of Tenofovir Alafenamide Fumarate and Comparison of Its Degradation and Stability Behaviour With Tenofovir Disoproxil Fumarate. J Pharm Biomed Anal. 2016;131:146–155.