Impurities Investigation of ARV-825 Proteolysis Targeting Chimera (PROTAC) Compound through Fraction Collection

Importance of the Topic

The emergence of proteolysis targeting chimera (PROTAC) technology represents a paradigm shift in drug discovery by harnessing the cell’s own ubiquitin–proteasome system to selectively degrade disease-related proteins. ARV-825 is a cutting-edge PROTAC targeting the bromodomain-containing protein BRD4, implicated in multiple cancers. Understanding its stability and degradation pathways is critical to ensure safety, efficacy and to guide formulation and storage strategies.

Objectives and Study Overview

This study aimed to isolate, identify and characterize the acidic degradation products of ARV-825 using both mass-directed and time-based fraction collection. Key goals included:

• Generating a forced degradation sample under defined acid stress.
• Fractionating degradation products via liquid chromatography coupled with mass-triggered and timed collection modes.
• Verifying purity and structural features of isolated fractions using orthogonal chromatographic methods and mass spectrometry.

Methodology and Instrumentation

Forced degradation was induced by treating a 1 mg/mL solution of ARV-825 in 45:55 acetonitrile–water with 0.5 M HCl for 2 hours at room temperature, followed by neutralization. Degradation mixtures were separated on a high-throughput UPLC-based system equipped with fraction collection.

• Chromatography system: Arc Premier with Binary Solvent Manager and Flow-Through Needle.
• Detectors: 2998 PDA and ACQUITY QDa II mass detector.
• Fraction collection: Waters Fraction Manager-Analytical (WFM-A) operated in mass-triggered and time-based modes.
• Columns: XSelect Premier CSH C18 (4.6 × 100 mm, 2.5 µm) and XSelect Premier HSS T3 (4.6 × 100 mm, 2.5 µm).
• Mass spectrometry: Electrospray positive ionization, m/z 200–1000, capillary 0.5 kV, probe temperature 600 °C.

Main Results and Discussion

Acid stress generated multiple degradation products, prominently at m/z 640, 472, 428 and 416. Mass-directed collection using targeted m/z triggers successfully isolated individual fractions, while time-based collection focused on the ARV-825 peak region improved resolution of closely eluting species. Orthogonal reanalysis on the HSS T3 column confirmed high purity of the major degradation product (m/z 640). Comparative analysis of day-1 and day-7 samples revealed increasing abundance of a minor species at m/z 463, indicating ongoing hydrolysis or rearrangement over time.

Benefits and Practical Applications

This workflow provides a robust platform for:

• Rapid isolation of low-level degradation products for structural elucidation.
• High-precision fraction collection reducing sample cross-contamination.
• Integration with automated software for streamlined method development in stability studies.

Future Trends and Potential Applications

Advances in PROTAC analysis may include:

• High-throughput screening of multiple stress conditions with automated fraction pooling.
• Coupling to high-resolution tandem mass spectrometry for detailed structural mapping.
• Application to a broader range of bifunctional modalities and complex biologics.
• Integration with machine-learning tools to predict degradation pathways and optimize linker design.

Conclusion

The combined use of mass-directed and time-based fraction collection with UPLC–MS detectors proved effective for isolating and characterizing ARV-825 degradation products. This approach enhances the analytical toolkit for PROTAC stability assessment, facilitating safer and more predictable drug development.

References

1. Rahman M, Marzullo B, Holman SW, Barrow M, Ray AD. Advancing PROTAC Characterization: Structural Insights through Adducts and Multimodal Tandem-MS Strategies. Journal of the American Society for Mass Spectrometry. 2024;35:285–299.
2. Liao X, Qian X, Zhang Z, et al. ARV-825 Demonstrates Antitumor Activity in Gastric Cancer via MYC-Targets and G2M-Checkpoint Signaling Pathways. Frontiers in Oncology. 2021;11:753119.
3. Berthelette KD, Collins C, Haynes K. Method Development of Proteolysis Targeting Chimera (PROTAC) Compound ARV-825 Forced Degradation Sample Using the Systematic Screening Protocol. Waters Application Note. 2024;720008328.