Multipass Ion Mobility Separation of GLP-1 Receptor Agonist Impurities Using the SELECT SERIES™ Cyclic™ IMS

Significance of the topic

Characterization of peptide impurities is critical for ensuring the safety, efficacy, and manufacturability of therapeutic peptides such as GLP-1 receptor agonists (e.g., semaglutide). Single-residue stereochemical variants (L→D substitutions) produce isomers with nearly identical chromatographic and mass properties, complicating detection and quantitation by LC-MS alone. Orthogonal separation methods that resolve these isomers rapidly and confidently are therefore essential for process development, quality control, and regulatory compliance.

Objectives and overview of the study

This application study evaluated the ability of multipass cyclic ion mobility spectrometry (Cyclic IMS) implemented on the SELECT SERIES Cyclic IMS platform to separate, identify, and quantify three D-amino-acid impurity isomers of semaglutide (D-Phe6, D-Ala19, D-Arg30) that co-elute under a standard 60-minute UPLC method. The work aimed to demonstrate scalable resolution via multiple mobility passes and targeted IMSn (slicing) workflows and to transfer the optimized mobility methods into LC-IM-MS analyses representative of formulation samples.

Methodology

Sample preparation and standards:

• Pure semaglutide API (all L) and synthetic standards of D-Phe6, D-Ala19, D-Arg30 were used.
• Direct-infusion: peptides dissolved to 1 mg/mL, diluted to 1 µM in water/acetonitrile with 0.1% formic acid for mobility optimization.
• LC-IM-MS: peptides prepared in 100 mM Tris-HCl pH 7.5, diluted to ~24.3 µM for injections.

Liquid chromatography:

• ACQUITY Premier UPLC using a ternary 60-minute gradient (water, acetonitrile, methanol) with 0.1% TFA.
• Column: ACQUITY Premier CSH C18, 2.1 × 150 mm, 1.7 µm, column temperature 60 °C.

Ion mobility / mass spectrometry strategy:

• SELECT SERIES Cyclic IMS platform operated in ESI+ mode; acquisition m/z 50–2000.
• Peptides produced primarily [M+3H]3+ and [M+4H]4+ ions; the [M+4H]4+ state provided superior mobility resolution and was the focus of separations.
• Multipass experiments scaled resolution by circulating ions for multiple passes (example: seven-pass and extended 18-pass experiments).
• Slicing IMSn workflow: isolate a mobility window after an initial separation, store the ions, then reinject the subset for additional passes to increase resolving power selectively.

Key instrument settings:

• Capillary voltage 2.50 kV; cone voltage 40 V; quadrupole profiles adjusted (examples provided in original method).
• Data acquired and processed with MassLynx and DriftScope software.

Used instrumentation

• SELECT SERIES Cyclic IMS (Waters) for multipass ion mobility separations.
• ACQUITY Premier UPLC System with Quaternary Solvent Manager.
• ACQUITY Premier CSH C18 Column, 2.1 × 150 mm, 1.7 µm.
• MassLynx and DriftScope software for acquisition and mobility data visualization.

Results and discussion

Direct infusion multipass mobility:

• For an equimolar mixture of the three D-residue isomers analyzed as [M+4H]4+, a seven-pass experiment produced a distribution with two closely spaced unresolved peaks (~50.40 ms and ~51.42 ms) and one nearly baseline-resolved peak (~52.27 ms). Identifications by individual standard analysis assigned the peaks as D-Arg30 (50.40 ms), D-Ala19 (51.42 ms), and D-Phe6 (52.27 ms).
• Increasing passive pass number beyond seven did not resolve the two closely spaced isomers further, indicating diminishing returns from uniform increases in path length.

Slicing IMSn targeted separation:

• To separate D-Arg30 and D-Ala19, a targeted slicing approach was used: after the seven-pass separation the mobility window containing the two unresolved species was isolated and stored in a pre-array, then reinjected and subjected to an additional 18 passes.
• This targeted reinjection yielded baseline separation of D-Arg30 and D-Ala19, demonstrating how IMSn with selective reinjection multiplies effective resolving power for a chosen subset of ions.

LC-IM-MS application to coeluting impurities:

• The optimized mobility functions were implemented in an LC-IM-MS method containing two channels: (a) a seven-pass channel to separate D-Phe6 and (b) a slicing/18-pass channel for the D-Arg30/D-Ala19 pair.
• Extraction of mobility-resolved chromatograms for the single LC peak (≈42 min) that contained all three impurities reproduced the infusion results, confirming that the multipass/slicing mobility strategy is compatible with chromatographic workflows and suitable for complex formulation matrices.

Interpretation:

• The [M+4H]4+ charge state displayed better mobility discrimination than the [M+3H]3+ state for these peptide isomers, emphasizing the role of charge state selection in mobility experiments.
• Slicing IMSn effectively concentrates resolving power where needed, avoiding unnecessarily long separations for the entire ion population while achieving baseline resolution for targeted isomers.

Benefits and practical applications

• Provides an orthogonal separation dimension to LC that can resolve stereoisomeric peptide impurities that co-elute chromatographically.
• Enables confident identification and quantitation of single-residue D-substitution impurities, improving impurity profiling for API development and QC release testing.
• IMSn slicing workflows offer targeted enhancement of resolution without extending chromatographic runtime or requiring specialized stationary phases.
• Transferability from direct infusion optimization to LC-IM-MS demonstrates applicability to real-world formulation samples and routine analyses.

Future trends and opportunities

• Integration of cyclic IMS with automated pass calculators and intelligent method scouting will streamline method development for complex peptide and protein therapeutics.
• Combination of high-resolution IMS with orthogonal fragmentation (IMSn with CID/ECD/ETD) can add structural confirmation for coeluting isomers, improving confidence in impurity assignments.
• Adoption in regulated environments will motivate development of standardized IMS performance metrics, calibration protocols, and validation practices for quantitative impurity assays.
• Broader application to other peptide therapeutics, synthetic peptide impurities, and post-translational variants (isobaric PTMs) where LC separation is insufficient.

Conclusion

Multipass cyclic ion mobility on the SELECT SERIES Cyclic IMS, combined with targeted slicing IMSn, resolves single-residue D-amino-acid isomers of semaglutide that are difficult to separate by LC-MS alone. The approach selectively amplifies mobility resolving power for targeted ion populations, translates to LC-IM-MS workflows, and supports robust impurity characterization for complex peptide therapeutic formulations.

References

1. Fioramonte M., Gomes A.F., Kass I.J., Barros F., Biffi R., Oliveira I.M. Multipass Ion Mobility Separation of GLP-1 Receptor Agonist Impurities Using the SELECT SERIES Cyclic IMS. Application Note. Waters Corporation; Published August 03, 2026.