PURIFICATION OF RADIOLIGAND PRECURSOR PSMA-617 FROM A FORCED DEGRADATION STUDY USING A WATERS MAXPEAK PREMIER OBD PREPARATIVE COLUMN

Purification of PSMA-617 from a Forced Degradation Mixture Using a MaxPeak Premier OBD Preparative Column — Executive Summary

Importance of the topic

Forced degradation studies and the subsequent isolation of degradation products are fundamental for characterizing the stability, degradation pathways, and impurity profiles of drug candidates and radioligand precursors. PSMA-617, a DOTA-containing radioligand precursor related to PLUVICTO®, presents specific chromatographic challenges because of metal-surface interactions (non-specific adsorption, NSA) and closely eluting degradants. Efficient analytical-to-preparative scale workflows that mitigate NSA and enable fast, high-purity isolation are essential in support of structural elucidation, reference standard production, and downstream radiopharmaceutical workflows where time and solvent consumption are critical.

Objectives and study overview

The study aimed to:

• Generate a forced degradation mixture of PSMA-617 under acidic, basic and oxidative stress to create a realistic impurity load.
• Develop and optimize focused UHPLC gradients to separate PSMA-617 from closely eluting degradants.
• Demonstrate predictable scale-up from analytical UHPLC to preparative isolation using MaxPeak Premier inert hardware and OBD preparative columns.
• Isolate and enrich target PSMA-617 and two closely eluting impurities, and assess the purity of collected fractions by UV and MS.

Methodology and experimental conditions

Sample generation and forced degradation:

• PSMA-617 stock: 5 mg/mL in water. Three 4 mL aliquots were treated separately with catalysts: 400 μL 1 N HCl (acidic), 400 μL 1 N NaOH (basic), and 400 μL 3% H2O2 (oxidative). Each vial was held at 70 °C for 24 h. The three stressed aliquots were combined for analysis and purification.

Mobile phases and general conditions:

• Mobile phase A: water with 0.1% formic acid. Mobile phase B: methanol with 0.1% formic acid.
• UV detection at 254 nm. Sample temperature ambient for analytical runs; column temperatures around 40–45 °C for analytical and preparative methods as indicated.

Gradient strategy and method development:

• Initial scouting: broad 5–95% B gradient to map components.
• Focused gradients: two iterations tried; the ultimate analytical focused gradient spanned 45–54% B (an 8% window) to resolve PSMA-617 from two closely eluting contaminants. A prior 51–59% B window failed to separate the target and adjacent peaks.
• Preparative gradient was adapted to match analytical retention using a similar L/dp ratio for scale transfer.

Chromatographic columns and scaling:

• Analytical column: XSelect Premier CSH Phenyl-Hexyl, 2.1 × 100 mm, 2.5 μm; flow 0.35 mL/min.
• Preparative column: XSelect Premier CSH Phenyl-Hexyl OBD (MaxPeak Premier inert hardware), 10 × 150 mm, 3.5 μm; flow ~5.67 mL/min; injection volumes scaled to 68 μL for prep vs 2–10 μL for analytical.
• L/dp ratios were maintained (≈40,000 for analytical and ≈42,857 for preparative) to support predictable chromatographic scale-up.

Used instrumentation

Analytical systems and detectors:

• ACQUITY UPLC H-Class System with Quaternary Solvent Manager, Sample Manager FTN-H, Column Manager, TUV detector and ACQUITY QDa mass detector (ESI+ for mass confirmation).

Preparative system:

• Waters AutoPurification System comprising a 2545 binary gradient module, 2767 sample manager, System Fluidics Organizer, 2998 photodiode array detector, Timberline column heater, and a 500 μL stainless-steel sample loop (for prep operations).
• MaxPeak Premier OBD preparative column constructed with inert hardware and HPS/MaxPeak surface technology to minimize NSA.

Key results and discussion

Chromatographic performance and separation:

• Scouting identified two contaminant peaks that eluted very close to PSMA-617 under the initial broad gradient. Focused gradient optimization was necessary to baseline-resolve the target from nearby degradants.
• The 45–54% B focused gradient successfully separated PSMA-617 from two small peaks that eluted immediately after the target; this gradient was translated to the preparative column with preserved selectivity and retention order.
• Use of MaxPeak Premier inert hardware and HPS surface technology reduced non-specific adsorption, which improved peak areas, reduced USP tailing factors, and narrowed peak widths compared to traditional metal-housed columns.

Preparative isolation and fraction analysis:

• The preparative 10 × 150 mm OBD column effectively resolved an impurity that eluted just before PSMA-617 and two distinct contaminants after the main peak. Peak collection targeted PSMA-617 and the two immediate impurities; a later eluting impurity was not collected.
• Mass spectrometry identification: PSMA-617 produced singly and doubly charged ions at m/z 1042 and 521. Several degradant peaks shared m/z values consistent with singly/doubly charged species at 1024/512, or other m/z values (371, 741) for coeluting degradants.
• Combined fraction pools from four preparative runs showed a single UV peak for the PSMA-617 pool and MS scans confirmed only the characteristic PSMA-617 ions, demonstrating high purity of the isolated target.
• The two impurity fraction pools were enriched (i.e., target-enriched but not fully pure). Chromatograms indicated that a polishing step using the same focused gradient would likely achieve pure isolates suitable for structural characterization.

Practical outcomes and operational efficiencies:

• Small-particle preparative columns and focused gradients enabled faster isolations with reduced solvent volumes and smaller fraction volumes, shortening dry-down times and supporting sustainability goals.
• Predictable scale-up (maintaining L/dp) simplified translation of analytical methods to preparative isolation workflows, which is valuable when rapid isolation is needed (e.g., radiochemistry workflows constrained by isotope half-life).

Benefits and practical applications of the method

• Minimized non-specific adsorption through inert column hardware improves recovery and fraction triggering reliability when handling metal-chelating analytes like DOTA-containing PSMA-617.
• Focused gradient design reduces coelution and enables clean collection of target and nearby impurities, supporting impurity enrichment and subsequent structural elucidation.
• Small particle prep columns (with preserved L/dp) provide high resolution at preparative scale, reducing solvent use and accelerating sample processing—advantageous for time-sensitive radiopharmaceutical production and standard generation.
• The approach supports a two-stage purification strategy: enrichment at prep scale followed by a polishing run to yield fully characterized impurities and standards.

Future trends and potential applications

Anticipated developments and broader uses include:

• Wider adoption of inert hardware and surface-passivated packing technologies to mitigate NSA for metal-chelating drugs and labile polar molecules.
• Integration of automated method scouting with predictive scale-up rules (L/dp matching) to accelerate transfer from analytical to preparative work with minimal trial-and-error.
• Enhanced sustainability in preparative chromatography via smaller-particle high-efficiency columns that reduce solvent consumption and fraction drying times.
• Application of the described workflow in radiopharmaceutical development pipelines where rapid isolation of precursors and degradants is necessary to support QA/QC, reference standard production, and degradation pathway elucidation.

Conclusions

The study demonstrates that combining focused gradient development, inert hardware (MaxPeak Premier HPS), and matched L/dp scaling enables reliable, high-purity isolation of PSMA-617 from forced degradation mixtures. The preparative MaxPeak Premier OBD column resolved critical impurities, allowed enrichment of minor degradants, and produced PSMA-617 fractions confirmed as pure by UV and MS. The approach reduces NSA-related artefacts, improves chromatographic performance (peak area, tailing, peak width), and yields operational efficiencies beneficial for purification labs—particularly in the radiopharmaceutical domain. A downstream polishing step is recommended to obtain fully pure degradants for structural identification.

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