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

Significance of the Topic

Proteolysis targeting chimeras (PROTACs) are an emerging class of bifunctional small molecules that harness the cellular ubiquitin–proteasome system to selectively degrade disease-related proteins. ARV-825 is a PROTAC that recruits cereblon E3 ligase to bromodomain-containing protein 4 (BRD4), showing promise in various cancer types. Comprehensive impurity profiling and stability evaluation of PROTACs are crucial for drug development, regulatory compliance, and ensuring therapeutic efficacy.

Objectives and Study Overview

This study aimed to isolate and characterize acid-induced degradation products of ARV-825 after forced degradation. By applying mass-directed and time-based fraction collection strategies, the workflow sought to capture specific degradation species based on their mass-to-charge ratios and to assess the stability of collected fractions over time.

Methodology and Instrumentation

• Sample Preparation: ARV-825 was stressed with 0.5 M HCl for two hours at room temperature, then neutralized and diluted to 1 mg/mL in 45:55 acetonitrile/water.
• Chromatography System: Waters Arc Premier equipped with Binary Solvent Manager, Flow-Through Needle, and Isocratic Solvent Manager.
• Columns: XSelect Premier CSH C18 (4.6×100 mm, 2.5 µm) and XSelect Premier HSS T3 (4.6×100 mm, 2.5 µm).
• Detection: ACQUITY QDa II mass detector (ESI+, 200–1000 m/z), 2998 PDA detector at 250 nm.
• Fraction Collection: Mass-triggered collection at m/z 640, 472, 428, and 416 using FractionLynx Application Manager; complementary time-based windows to isolate closely eluting peaks.
• Data Control: Empower Chromatography Data System and MassLynx Software v4.2.

Key Results and Discussion

Acid stress generated multiple degradation products, notably ions at m/z 640, 472, 428, and 416. Mass-directed collection successfully isolated these species into single or pooled fractions, enhancing sample throughput. Orthogonal analysis on the HSS T3 column confirmed the chromatographic and spectral purity of the m/z 640 species. Time-based fractionation around the parent ARV-825 peak demonstrated precise capture of coeluting impurities. Monitoring of fraction solutions over seven days revealed the emergence of an additional degradation ion at m/z 463, indicating ongoing in-solution degradation.

Benefits and Practical Applications

• Enables targeted isolation of low-abundance degradation products in complex PROTAC formulations.
• Improves sensitivity and specificity by combining mass-triggered collection with orthogonal separation.
• Automates fractionation to reduce manual intervention and improve reproducibility.
• Supports regulatory submissions by providing detailed impurity profiles and stability data.

Future Trends and Opportunities

The growing use of PROTACs will drive demand for advanced impurity profiling workflows. Integration of high-resolution MS and adaptive fractionation triggers could enhance separation of isomeric species. Real-time data-driven collection algorithms may streamline isolation of labile intermediates. Further coupling with MS/MS and NMR for structural elucidation will elucidate detailed degradation pathways. The described approach can be extended to other targeted degrader modalities and complex biologics.

Conclusion

This work presents an efficient and robust strategy for isolating and characterizing ARV-825 degradation products using mass-directed and time-based fraction collection. The combination of targeted MS triggers, orthogonal chromatography, and automated fraction management provides a comprehensive platform for PROTAC impurity analysis.

References

1. Rahman M, Marzullo B, Holman SW, Barrow M, Ray AD. Advancing PROTAC Characterization: Structural Insights through Adducts and Multimodal Tandem-MS Strategies. J Am Soc Mass Spectrom. 2024;35:285-299.
2. Liao X, Qian X, Zhang Z, Tao Y, Li Z, Zhang Q, Liang H, Li X, Xie Y, Zhuo R, Chen Y, Jiang Y, Cao H, Niu J, Xue C, Ni J, Pan J, Cui D. ARV-825 Demonstrates Antitumor Activity in Gastric Cancer via MYC-Targets and G2M-Checkpoint Signaling Pathways. Front Oncol. 2021;11:753119.
3. Berthelette KD, Collins C, Haynes K. Method Development of PROTAC Compound ARV-825 Forced Degradation Sample Using Systematic Screening Protocol. Waters Application Note 720008328. 2024.