Predictable Isolation of Radioligand Therapy Precursor PSMA-617 and Closely Eluting Impurities from a Forced Degradation Study Using a MaxPeak™ Premier OBD™ Preparative Column

Significance of the topic

Radioligand therapy precursors such as PSMA-617 require rigorous characterization and access to high-purity material for analytical reference standards and downstream radiolabeling. Because PSMA-617 contains metal-chelating functionality, it is prone to non-specific adsorption to metal surfaces, complicating both analysis and preparative isolation. Reliable analytical-to-preparative scale translation and inert-column technologies therefore play a critical role in producing pure precursor material rapidly and predictably — an important practical requirement given the time constraints imposed by radionuclide half-lives in radiopharmaceutical workflows.

Objectives and study overview

The study aimed to (1) generate a stressed degradation mixture of PSMA-617 at preparative scale, (2) optimize an analytical UPLC method to resolve PSMA-617 from closely eluting low-level degradants, and (3) demonstrate predictable scale-up to preparative isolation using an inert-surface MaxPeak Premier OBD preparative column on a UV-directed AutoPurification platform. The ultimate goal was to recover PSMA-617 with high purity suitable for use as a reference standard and to evaluate the enrichment of neighboring impurities for potential polishing steps and structural characterization.

Methodology and experimental design

Forced-degradation sample preparation:

• Stock: PSMA-617 at 5 mg/mL in water.
• Three 4 mL aliquots placed in polypropylene vials; 400 µL of a catalyst added to each vial, then 400 µL of stressor: 1 N HCl (acid), 1 N NaOH (base), or 3% H2O2 (oxidative).
• Stress conditions: 70 °C for 24 h for each condition.
• After treatment, all aliquots were combined and stored prior to analysis and purification.

Analytical development and scale-up strategy:

• Initial scouting gradient (5–95% B) identified at least two small contaminants eluting adjacent to PSMA-617.
• Focused gradient development narrowed the organic composition window (final optimized 45–54% B) to resolve the main peak from immediate neighbors.
• Direct scaling was implemented preserving the column length/particle diameter (L/dp) ratio by moving from a 2.1 × 100 mm, 2.5 µm analytical XSelect CSH Phenyl-Hexyl column (L/dp ≈ 40,000) to a custom 10 × 150 mm, 3.5 µm MaxPeak Premier OBD prep column (L/dp ≈ 42,857).
• Fractionation was controlled by UV-directed AutoPurification with mass confirmation via ESI positive mode to identify collected peaks.

Used instrumentation

Key instruments and consumables used in the study included:

• Waters AutoPurification System with 2998 PDA detector and FractionLynx control for fraction collection.
• ACQUITY UPLC H-Class System with ACQUITY TUV detector and QDa mass detector for analytical monitoring and peak identification.
• Columns: XSelect Premier CSH Phenyl-Hexyl, 2.1 × 100 mm, 2.5 µm (analytical) and MaxPeak Premier XSelect CSH Phenyl-Hexyl OBD preparative column, 10 × 150 mm, 3.5 µm (custom prep).
• Column temperature control via Timberline column heater; typical column temperatures: analytical ~40 °C, preparative ~45 °C.
• Mobile phases: A = water + 0.1% formic acid; B = methanol + 0.1% formic acid.
• Typical flows and injections: analytical flow 0.35 mL/min (2–10 µL injections); preparative flow 5.67 mL/min with 68 µL injections; prep sample loop 500 µL (stainless steel).
• Data handling: MassLynx v4.2 and Empower 3 for acquisition and processing.

Main results and discussion

Analytical scouting identified multiple degradants closely flanking the PSMA-617 peak. A focused gradient window (45–54% B) substantially improved resolution, enabling baseline separation of the main peak from the nearest impurities at analytical scale. Maintaining a comparable L/dp during scale-up allowed the preparative OBD column to reproduce the analytical retention profile and selectivity, facilitating straightforward fraction assignment.

Mass spectrometric verification (ESI+) confirmed that the principal preparative peak corresponded to PSMA-617 (singly charged m/z ≈ 1042 and doubly charged m/z ≈ 521). The neighboring impurity peaks showed distinct m/z values, with several species sharing m/z ≈ 1024/512, indicating related degradant families. Fraction collection parameters were tuned to avoid co‑collection of the impurity eluting immediately before PSMA-617; as a result, pooled PSMA-617 fractions were shown by UV and MS to be free of the closely eluting contaminants.

Impurity pools collected from preparative runs were enriched but not fully pure; their chromatograms indicated that a subsequent polishing isolation using the same focused gradient would likely yield pure material for full structural elucidation. Enrichment increases yield for downstream workup and characterization.

Benefits and practical applications

The workflow demonstrated several practical advantages:

• Inert hardware (High Performance Surface technology) reduced non-specific adsorption of the chelating PSMA-617, improving peak area, symmetry, and reproducibility.
• Direct analytical-to-preparative scalability (matched L/dp and OBD packing control) delivered predictable retention and selectivity when moving to prep scale.
• Small-particle preparative packing (3.5 µm) enhanced resolution of closely eluting impurities while enabling lower-volume fraction collection and faster solvent evaporation.
• Improved peak shape and detection aided precise UV-triggered fractionation, minimizing cross-contamination and accelerating purification throughput.
• Elimination of lengthy column conditioning for NSA minimized downtime before purification runs.

Practically, this approach produces high-purity PSMA-617 suitable as a reference standard and supports efficient impurity enrichment for identification — a workflow well aligned with needs in radiopharmaceutical development, where speed and material quality are critical.

Future trends and potential applications

Expected developments and opportunities include:

• Broader adoption of inert-surface preparative columns across radiopharmaceutical purification to mitigate metal-binding artefacts.
• Integration of higher-resolution small-particle preparative media with automated fractionation for rapid generation of reference standards and impurity libraries.
• Application of iterative enrichment/polishing workflows (multi-stage prep runs) to isolate and structurally characterize low-level degradants encountered in forced-degradation studies.
• Use of rapid preparative methods to shorten timelines in radiopharmaceutical development, supporting activities constrained by radionuclide decay.

Conclusion

By combining inert-surface (HPS) stationary phases, carefully focused gradients, and a controlled prep packing process (MaxPeak Premier OBD columns), the study achieved predictable analytical-to-preparative scale translation and isolated PSMA-617 with high purity from a forced-degradation mixture. The preparative column reproduced analytical selectivity, enabling clear separation of closely eluting impurities and efficient fraction collection. Impurity pools were enriched sufficiently to support subsequent polishing and structural elucidation. Overall, the coordinated use of stationary-phase chemistry, inert hardware, and system-level fraction control provides an effective strategy for purification tasks in radiopharmaceutical development.

References

1. Giugliano F, et al. Cancer Treatment Reviews 2025;136:102940.
2. Berthelette K, Aiello M, Collins C, Walter TH. Development of a UPLC Method for a Forced Degradation Study of Radioligand Therapy Precursor PSMA-617. Waters Application Note; 2025.
3. DeLano M, et al. Analytical Chemistry 2021;93:5773–5781. DOI:10.1021/acs.analchem.0c05203.