Method Development of Proteolysis Targeting Chimera (PROTAC) Compound ARV-825 Forced Degradation Sample Using the Systematic Screening Protocol

Significance of the Topic

Proteolysis Targeting Chimeras (PROTACs) represent a novel therapeutic strategy by inducing selective protein degradation, offering advantages over traditional inhibitors. ARV-825, a PROTAC targeting BRD4, shows promise in treating various cancers. Understanding its forced degradation behavior is crucial for assessing stability, ensuring patient safety, and guiding formulation development.

Objectives and Study Overview

This study aimed to develop a robust UPLC-MS method to characterize degradation products of ARV-825 under accelerated stress conditions. A systematic screening protocol was applied to expedite method development and achieve baseline resolution of ARV-825 and its degradants.

Methodology

A combined forced degradation sample was prepared by subjecting ARV-825 to acid, base, thermal, and oxidative stress at 70 °C. Photolytic degradation was evaluated but showed no significant change. Degradation mixtures were quenched and pooled for analysis.

Used Instrumentation

UPLC system and detectors:

• ACQUITY UPLC H-Class Plus with Quaternary Solvent Manager and Sample Manager FTN
• ACQUITY QDa Mass Detector (ESI+ full-scan and SIR)

Columns screened (MaxPeak Premier HPS hardware):

• BEH C18
• CSH Phenyl-Hexyl
• HSS PFP
• HSS T3
• BEH Shield RP18
• BEH C18 AX

Mobile phases:

• Water with formic acid
• Acetonitrile or methanol

Data acquired with Empower 3 CDS.

Main Results and Discussion

pH screening identified low-pH formic acid conditions as optimal for retaining degradants. Column screening with acetonitrile revealed the HSS T3 phase as providing the best separation profile. Gradient optimization (0–70% B in 5.3 min) improved retention of early-eluting degradants and reduced cycle time. Comparison of MaxPeak Premier HPS hardware versus standard stainless-steel columns demonstrated superior peak shape, selectivity, and reproducibility with HPS technology due to minimized metal-analyte interactions.

Benefits and Practical Applications

• Accelerated method development through a tiered systematic protocol.
• Baseline resolution of all ARV-825 degradants and main peak.
• Enhanced reproducibility and sensitivity using MaxPeak Premier HPS columns.

Future Trends and Opportunities

Integration of high-throughput automated screening and novel stationary phase chemistries will further reduce development timelines. Advances in hybrid surface treatments and microfluidic LC-MS systems may enhance trace-level degradant detection and expand applicability to complex biologics.

Conclusion

Applying a systematic screening workflow combined with MaxPeak HPS technology enabled rapid development of a stability-indicating UPLC-MS method for ARV-825 forced degradation samples. The approach ensures reliable degradant identification and robust performance, supporting critical stability assessment in PROTAC drug development.

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