Analysis of PROTAC Molecule Bavdegalutamide (ARV-110) Using LC/UV and LC/MS

Analysis of the PROTAC Molecule Bavdegalutamide (ARV-110) by LC/UV and LC/MS — Summary

Significance of the topic:
Bifunctional PROTAC molecules such as bavdegalutamide (ARV-110) represent a new therapeutic modality that induces target protein degradation rather than simply inhibiting function. Their structural complexity, multiple synthetic steps and multifunctional moieties increase the risk of closely related synthesis impurities and diverse degradation products. Robust chromatographic separation combined with high-resolution mass spectrometry is therefore essential to characterize identity, monitor stability, support process development and meet regulatory requirements for impurity profiling and forced‑degradation studies.

Objectives and study overview:

• Develop and compare LC methods to separate ARV-110 from synthesis-related and degradation-derived impurities.
• Perform forced degradation (oxidative, acidic, basic and thermal) to generate potential degradants and evaluate stability.
• Use high-resolution LC/Q-TOF MS/MS and molecular-structure software to assign accurate masses, map fragmentation and propose structures for major impurities.

Methodology:

• Sample preparation: ARV-110 stock in DMSO (5 mg/mL) with working dilutions for LC/UV and LC/MS analyses. Forced degradation conditions included: 10% H2O2 at room temperature for 3 h (oxidative), 1 N HCl at 80 °C for 1 h (acid hydrolysis), 1 N NaOH at room temperature for 1 h (base hydrolysis), and dry heat at 70 °C for 3 h (thermal).
• Chromatography: Reversed‑phase gradients using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); 12 min total run time, 0.4 mL/min flow, 50 °C column compartment, and small injection volumes (0.5 µL for MS) to accommodate narrow peaks and high-efficiency columns.
• Column screening: Multiple superficially porous and hybrid stationary phases were evaluated to optimize selectivity for PROTAC impurities (C18 variants, pentafluorophenyl, phenyl‑hexyl and a peptide‑optimized hybrid phase).

Used instrumentation:

• LC: Agilent 1290 Infinity II platform with high-speed pump, multisampler, multicolumn thermostat and diode array detector.
• Columns tested: Agilent AdvanceBio Peptide Plus (2.1 × 50 mm, 2.7 µm), Poroshell 120 PFP, Poroshell 120 EC‑C18, Poroshell 120 Aq‑C18 and Poroshell 120 Phenyl‑Hexyl (all 2.1 × 50 mm formats).
• MS: Agilent Revident LC/Q‑TOF (G6575A) operated in positive ESI; acquisition m/z range ~100–1700, high-resolution MS and MS/MS with narrow isolation, active exclusion and fast acquisition rates to capture chromatographic peaks.
• Software: Agilent OpenLab CDS, MassHunter acquisition and qualitative tools, and Molecular Structure Correlator (MSC) for automated fragment-to-structure correlation.

Main results and discussion:

• Column selection: The AdvanceBio Peptide Plus column provided the best overall selectivity and peak shape for ARV-110 and its related substances compared with the tested Poroshell phases. Its charged-hybrid/C18 surface reduced band broadening and improved resolution of closely eluting impurities, an important outcome given the structural similarity among potential byproducts and positional isomers.
• Forced-degradation outcomes: Oxidative stress (H2O2) generated multiple degradants (eight distinct peaks observed by LC/UV), indicating susceptibility of certain moieties (e.g., piperazine or cyclohexylamine-like fragments). Thermal stress produced no new significant degradants under the conditions tested, showing thermal robustness. Acidic hydrolysis yielded fewer products, while base hydrolysis resulted in complete loss of the parent ARV-110 peak and appearance of early-eluting, highly polar products consistent with extensive hydrolysis.
• High-resolution MS characterization: LC/Q‑TOF detected both singly and doubly charged parent ions for ARV‑110 and provided mass accuracy within ±1 ppm for measured ions and fragments, enabling confident molecular formula assignments. MS/MS fragmentation mapping of ARV‑110 produced a set of diagnostic product ions that supported structural confirmation of the parent and aided interpretation of impurity structures.
• Major impurity identification: The dominant impurity at m/z 470.1947 was isolated by LC/MS and subjected to MS/MS. MSC software used accurate precursor and fragment masses to propose candidate structures; the top-scoring proposal (correlation ≈ 97.7%) matched an impurity reported in prior literature/patent sources, supporting the identification. Additional minor unknowns were annotated with tentative formulas and structural features, though unambiguous assignments for low-abundance species remain challenging without reference standards.

Benefits and practical applications of the method:

• The combined strategy of column screening and high-resolution LC/Q‑TOF MS/MS enables sensitive detection and confident characterization of PROTAC-related impurities and degradants, supporting process optimization and impurity control.
• Accurate-mass MS and automated fragmentation correlation (MSC) accelerate structure elucidation workflows and reduce time to identify major impurities when reference standards are unavailable.
• The demonstrated stability profiles inform formulation and storage strategies; for example, susceptibility to oxidative and basic conditions highlights the need for antioxidant considerations and careful pH control during processing and formulation.
• Method features (short run time, high selectivity) are compatible with routine QC screening, forced‑degradation testing for regulatory submissions and support of research-stage impurity profiling.

Future trends and potential uses:

• Broader adoption of high-resolution MS with automated structure-correlation tools will continue to speed impurity identification for complex modalities like PROTACs.
• Integration of orthogonal separation techniques (ion-mobility, two-dimensional LC) and complementary detectors (native/structural MS, UV/charged aerosol) will improve characterization of isomeric and highly polar degradants.
• Increased use of in-silico fragmentation, machine-learning assisted spectral interpretation and standardized impurity databases will further reduce ambiguity for low‑abundance unknowns.
• Regulatory expectations will likely push for standardized forced-degradation workflows tailored to bifunctional molecules, and analytical platforms that combine throughput with high structural confidence will be preferred.

Conclusion:
A targeted workflow combining chromatographic column screening and high-resolution LC/Q‑TOF MS/MS reliably separated and characterized ARV‑110 and its degradation products. The AdvanceBio Peptide Plus stationary phase offered superior selectivity for this PROTAC, while Q‑TOF accuracy and MSC-assisted MS/MS interpretation enabled confident assignment of a major impurity (m/z 470.1947). This approach provides a practical path for impurity profiling, stability assessment and structure elucidation that supports development and regulatory readiness for complex therapeutic modalities.

References:

• Anaya YA; et al. Proteolysis‑Targeting Chimeras in Cancer Therapy: Targeted Protein Degradation for Next‑Generation Treatment. Cancer 2025, 131(21):e70132.
• Snyder LB; et al. Preclinical Evaluation of Bavdegalutamide (ARV‑110), a Novel PROteolysis TArgeting Chimera Androgen Receptor Degrader. Molecular Cancer Therapeutics 2025, 24(4), 511–522.
• Moreau K; et al. Proteolysis‑Targeting Chimeras in Drug Development: A Safety Perspective. British Journal of Pharmacology 2020, 177(8), 1709–1718.
• ICH Expert Working Group. Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products Q5C. ICH Harmonized Tripartite Guideline (1995).
• Methods of Manufacturing a Bifunctional Compound, Ultrapure Forms of the Bifunctional Compound, and Dosage Forms Comprising the Same. US Patent 12,043,612 B2, July 23, 2024.