Separating and Identifying Site-Specific Isomeric Amino Acids in GLP-1 Receptor Agonists on the Cyclic™ IMS P20 Mass Spectrometer

Separating and Identifying Site-Specific Isomeric Amino Acids in GLP-1 Receptor Agonists — Executive Summary

Significance of the topic

Isomeric amino acid variants (for example D-amino acid incorporation or isoaspartate formation) are challenging impurities in peptide therapeutics because they do not change peptide mass and frequently co-elute in reversed-phase LC. Such stereochemical changes can alter peptide conformation, stability, potency, and immunogenicity, making confident detection and site localization essential for drug substance and product characterization, stability studies, and regulatory compliance.

Objectives and overview of the study

This application note demonstrates an integrated LC–ion mobility–MS workflow to detect, separate, and localize site-specific isomeric amino acid impurities in the GLP-1 receptor agonist liraglutide. The main aims were: to resolve coeluting isomeric forms invisible to LC-MS alone using high-resolution cyclic ion mobility (IMS); to localize the site(s) of stereochemical modification via product ion-level mobility after pre-IMS fragmentation; and to improve sensitivity for low-abundance fragments using Wideband Enhancement (WBE).

Methodology and used instrumentation

The experimental approach combined reversed-phase UPLC separation with high-resolution cyclic IMS and MS/MS performed upstream of the mobility separator. Key elements of the methodology:

• Sample: Liraglutide standard prepared at 10 μg/mL and analyzed from low‑binding vials to minimize sample loss.
• Chromatography: ACQUITY Premier UPLC system with a Peptide BEH C18 2.1 × 100 mm, 1.7 μm column, 45 °C column temperature, formic acid/acetonitrile mobile phases and gradients extended to improve chromatographic resolution where required.
• Mass spectrometry and IMS: Waters Cyclic IMS P20 Mass Spectrometer operated in positive mode, sensitivity setting, mass range 50–4000 m/z. The cyclic IMS device enabled single-pass and multi-pass (up to eight passes) mobility separations to increase resolving power.
• Fragmentation and acquisition: True MS/MS upstream of the IMS device using CID and optional ECD to produce b/y and c/z fragments. Wideband Enhancement (WBE) acquisition mode was used to boost product-ion signal intensity across a broad m/z range.

Used instrumentation

• Waters ACQUITY Premier UPLC System (binary)
• ACQUITY UPLC Peptide BEH C18 Column, 130 Å, 1.7 μm, 2.1 × 100 mm
• Waters Cyclic IMS P20 Mass Spectrometer with pre‑IMS fragmentation capability (quadrupole positioned upstream of cyclic mobility device)
• QuanRecovery vials with MaxPeak HPS surfaces to reduce adsorption

Main results and discussion

Key experimental findings and interpretation:

• Detection and resolution of multiple isomers: LC–MS of liraglutide revealed a minor, earlier‑eluting isomeric component. Single‑pass IMS separation resolved this feature into two distinct mobility species (Iso1 and Iso2) present at ~0.24% and ~0.61%. By increasing mobility resolving power via eight passes of the cyclic IMS device, a third, very low‑abundance species (Iso3, ~0.09%) was uncovered that was not visible in single‑pass data, even with an extended LC gradient.
• Pre‑IMS fragmentation enables residue localization: Performing MS/MS upstream of the mobility device produced product ions that were separated by IMS. Comparison of arrival time distributions (ATDs) for matched product ions between the main peptide and each isomer allowed localization of stereochemical differences: Iso1 differences were traced to serine 8 and Iso2 to serine 11. The logic follows that product ions containing the modified residue exhibit altered mobility behavior relative to matching fragments from the main species.
• WBE boosts confidence for low‑abundance fragments: Many product ions from low‑level isomers suffer from poor signal and noisy ATDs. Implementing WBE increased product ion intensities (reported ~6‑fold for a representative ion), improving peak shape and isotopic fidelity and enabling reliable ATD comparisons for localization.
• Orthogonal fragmentation modes: Using both CID (b/y ions) and ECD (c/z ions) prior to IMS provided complementary fragment series, increasing the robustness of residue localization by corroborating ATD shifts across different fragmentation chemistries.

Benefits and practical applications

This IMS‑enabled workflow provides several practical advantages for peptide therapeutic analysis:

• Enhanced impurity detection—multipass cyclic IMS finds low‑abundance isomers that standard LC‑MS may miss, improving impurity profiling completeness.
• Residue-level localization—pre‑IMS fragmentation with ATD comparison allows site-specific assignment of stereochemical changes without relying solely on chromatographic separation or mass shifts.
• Improved sensitivity and data quality—WBE increases detectability and ATD fidelity for low‑intensity fragments, supporting confident structural assignments.
• Streamlined follow-up—localized assignment of modifications can reduce the need for extensive orthogonal experiments, accelerating characterization workflows for synthesis optimization, stability testing, and release testing.

Future trends and potential applications

Potential directions and broader uses informed by this study:

• Wider adoption of high‑resolution cyclic IMS for characterization of other peptide modalities (e.g., long peptides, therapeutic proteins with isomeric PTMs) and synthetic impurities.
• Integration of IMS‑based fragment localization with automated data analysis tools and databases to accelerate routine identification of isomeric impurities and support regulatory submissions.
• Combination with orthogonal structural methods (NMR, hydrogen/deuterium exchange, or targeted enzymatic assays) for definitive stereochemical confirmation where required.
• Further improvements in acquisition modes and ion optics to increase throughput while retaining high resolving power for routine QA/QC environments.

Conclusion

The combined LC–cyclic IMS–MS workflow demonstrated here enables detection and residue‑level localization of isomeric amino acid impurities in a GLP‑1RA model compound. Multipass cyclic IMS increases separation power to reveal low‑level isomers, pre‑IMS fragmentation localizes changes to specific residues (serine 8 and serine 11 in the liraglutide example), and WBE enhances fragment signal to make ATD comparisons reliable. Collectively, these capabilities improve confidence in impurity profiling for peptide therapeutics and reduce ambiguity that arises from mass‑equivalent isomeric variants.

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