Degradation Products of ARV-825 Proteolysis Targeting Chimera (PROTAC) Compound: Isolation and Purification Using Analytical Scale Fraction Collection

Importance of the Topic

Proteolysis targeting chimeras (PROTACs) represent an emerging class of therapeutic agents that harness the cell’s own degradation machinery to eliminate disease-driving proteins. ARV-825 is a novel PROTAC designed to induce selective degradation of bromodomain-containing protein 4 (BRD4), implicated in multiple cancers. Understanding degradation pathways and impurity profiles of PROTACs under stress conditions is essential for developing stability-indicating methods, ensuring drug safety, and guiding formulation strategies.

Objectives and Study Overview

This application note investigates acid-induced degradation products of ARV-825. The primary goals were to generate forced-degradation samples via hydrochloric acid treatment, isolate major degradants at analytical scale, and verify the purity of collected fractions. The study demonstrates the combination of chromatographic separation with mass-directed fraction collection to recover individual degradation products for subsequent characterization.

Methodology and Instrumentation

Forced degradation conditions:

• ARV-825 at 1 mg/mL in 45:55 acetonitrile/water
• Treatment with 0.5 M HCl for 2 h at ambient temperature, followed by neutralization with 0.5 M NaOH

Chromatography and fraction collection:

• Arc™ Premier LC System with Binary Solvent Manager and Flow-Through Needle
• Waters™ Fraction Manager-Analytical (WFM-A) with low-dispersion flow path
• XSelect™ Premier CSH™ C18 column (4.6 × 100 mm, 2.5 µm)
• UV detection (2998 PDA, 250 nm) and ACQUITY™ QDa™ II Mass Detector (ESI+, m/z 200–1000)
• MassLynx™ Software with FractionLynx™ for automated, mass-triggered or time-based fraction collection

Key parameters:

• Injection volume: 40 µL
• Column temperature: 30 °C; sample temperature: 15 °C
• Gradient elution with 0.1% formic acid in water and acetonitrile
• ISM makeup solvent (50:50 acetonitrile/water, 0.1% formic acid) for enhanced ionization in MS

Main Results and Discussion

The forced degradation yielded multiple ARV-825 degradants, which were baseline separated on the CSH C18 column. Mass-directed fraction collection using target m/z values (640, 472, 428, 416) enabled selective recovery of each product. Pooling of multiple collections per vial increased sample yield without compromising purity.

Orthogonal re-analysis using an XSelect HSS T3 column and Empower™ CDS confirmed high chromatographic purity (>97%) and spectral homogeneity for major fractions. One degradant (m/z 472) exhibited secondary degradation during storage, highlighting the need for rapid analysis and appropriate handling of isolated compounds.

Time-based fractionation across the ARV-825 peak region, followed by focused gradient re-injection, resolved coeluting components (m/z 923 and 462) and illustrated the WFM-A’s ability to slice closely eluting peaks.

Benefits and Practical Applications

• High-resolution, low-dispersion fraction collection at analytical scale preserves limited sample quantities.
• Mass-triggered isolation accelerates identification of targeted degradants.
• Automated workflows in MassLynx™ with FractionLynx™ improve traceability and reproducibility.
• Orthogonal confirmation ensures structural integrity of isolated products for downstream characterization.

Future Trends and Opportunities

Advancements in PROTAC development will drive demand for robust stress-testing and impurity profiling methods. Integration of high-resolution MS/MS and ion mobility could further characterize complex degradation pathways. Miniaturized fraction collectors and microflow LC platforms may enhance sensitivity and reduce solvent consumption. Coupling automated fractionation with rapid structural elucidation tools (e.g., NMR, high-resolution MS) promises streamlined workflows for next-generation therapeutic candidates.

Conclusion

The combined use of the Arc™ Premier System, WFM-A, and ACQUITY™ QDa™ II Mass Detector enables efficient, high-purity isolation of ARV-825 degradation products under forced-degradation conditions. Automated mass-directed and time-based fraction collection, coupled with orthogonal re-analysis, provides a powerful approach for stability-indicating method development in PROTAC drug discovery.

Reference

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. Liu Z, Hu M, Yang Y, et al. An Overview of PROTACs: A Promising Drug Discovery Paradigm. Mol Biomed. 2022;3:46.
3. Liao X, Qian X, Zhang Z, et al. ARV-825 Demonstrates Antitumor Activity in Gastric Cancer via MYC-Targets. Front Oncol. 2021;11:753119.
4. Li Z, Lim SL, Tao Y, et al. PROTAC Bromodomain Inhibitor ARV-825 Displays Anti-Tumor Activity in Neuroblastoma. Front Oncol. 2020;10:574525.
5. He L, Chen C, Gao G, Xu K, Ma Z. ARV–825 Induced BRD4 Protein Degradation as a Therapy for Thyroid Carcinoma. AGING. 2020;12(5).
6. ICH Q1A. Stability Testing of New Drug Substances and Products. ICH; 2003.
7. Waters™ Fraction Manager-Analytical. Waters Corporation; 2025.
8. Berthelette KD, Collins C, Haynes K. Method Development of PROTAC Compound ARV-825 Forced Degradation Using Systematic Screening Protocol. Waters Application Note. 2024.