LC-UV/MS Workflows Enabling Orthogonal Impurity Profiling of GLP-1 Analogs

Importance of the topic

Peptide therapeutics such as GLP-1 receptor agonists (example: exenatide) are chemically complex and prone to sequence variants and spontaneous post‑translational changes (e.g., deamidation, amino‑acid insertions). Robust impurity profiling is critical for safety, potency and regulatory compliance. Combining orthogonal liquid chromatography selectivity with mass spectrometry (LC‑UV/MS) increases detection power for impurities that may be invisible or coelute in a single chromatographic mode, improving raw material screening and quality control workflows.

Objectives and study overview

This work demonstrates QC‑friendly LC‑UV/MS workflows that employ complementary reversed‑phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations to reveal orthogonal impurity profiles of GLP‑1 analogs, using exenatide as a model compound. Goals included: detect sequence variants and deamidation products, evaluate column chemistries and mobile phase variables that control separation, and show how MS complements UV detection to discover co‑eluting impurities.

Methodology

Samples: exenatide raw material and synthesized standards representing deamidation species and an insertion impurity (exenatide + proline). Forced degradation (high‑pH, 37 °C overnight) and spike‑in experiments were used to generate and test impurity separation.

Chromatographic screening:

• Reversed‑phase chromatography on ACQUITY Premier Peptide CSH C18 (130 Å, 1.7 µm; 2.1 × 100 mm), elevated temperature (60 °C) and formic‑acid/ACN mobile phases.
• HILIC using Waters BEH HILIC and BEH Amide stationary phases (130 Å; 1.7 µm; 2.1 × 100 mm and 2.1 × 150 mm), mobile phases with varied buffer type, ionic strength and pH (formate/acetate, NH4HCO2, NH4CH3O2), and organic content (ACN).

MS complement: low‑to‑high resolution MS was applied—ACQUITY QDa II Mass Detector for LC‑compatible screening and Waters Xevo QTof G3 with UNIFI for sequence confirmation. MS acquisition parameters (example QDa II): positive ESI, 250–1500 m/z, 5 Hz scan, capillary 1.5 kV, cone 15 V, probe 600 °C.

Method scouting variables evaluated:

• pH effects on retention (acidic pH reduced retention due to increased positive charge and reduced electrostatic interactions).
• Ionic strength effects (higher salt masked stationary phase charge sites, decreasing retention).
• Ion‑pairing reagents (TFA, DFA, formic acid) to modulate elution order and ion‑pair strength.
• Column chemistry and gradient slope to optimize resolution and throughput.

Used instrumentation

• LC: ACQUITY Premier System.
• Detectors: ACQUITY QDa II Mass Detector; Waters Xevo QTof G3 Mass Spectrometer (for sequence confirmation) with UNIFI Scientific Information System.
• Columns: ACQUITY Premier BEH HILIC Column (130 Å, 1.7 µm, 2.1 × 100 mm), ACQUITY Premier BEH Amide Column (130 Å, 1.7 µm, 2.1 × 150 mm), ACQUITY Premier Peptide CSH C18 Column (130 Å, 1.7 µm, 2.1 × 100 mm).
• Software: Empower 3.8.1; UNIFI for high‑resolution MS analysis.

Main results and discussion

1) Co‑elution in RP: Under RP conditions, a sequence‑insertion impurity (exenatide + proline, Δ+97.1 Da) coeluted with the native peptide in UV chromatograms. MS revealed the impurity despite lack of UV resolution. Lowering RP column temperature improved separation marginally but pushed beyond robust operating ranges.

2) HILIC resolves challenging impurities: BEH HILIC and BEH Amide phases provided baseline separation of the insertion impurity and improved resolution of deamidation species. Example: BEH HILIC achieved baseline resolution with Rs ~2.94 for the insertion impurity, and BEH Amide improved peak symmetry and throughput (faster gradients while maintaining separation).

3) Sensitivity to mobile phase composition: Retention and elution order in HILIC were strongly dependent on buffer pH, ionic strength, and the choice of counter‑ion/ion‑pairing reagent. At constant ionic strength, lower pH reduced retention by increasing positive charge on peptide and stationary phase, weakening electrostatic attraction. Higher salt concentrations masked stationary phase charges and reduced retention. Ion‑pairing reagents (TFA, DFA, formiate) changed elution order of deamidation species due to differing anion hydrophobicity and pKa values.

4) Deamidation detection: Deamidation introduces a +1 Da mass shift per event, challenging to detect by UV or low‑resolution MS alone. Synthesized deamidation standards and high‑resolution MS enabled unambiguous identification and allowed chromatographic evaluation of these species. Forced degradation produced >12% total impurities, highlighting degradation risk under stress conditions.

5) Orthogonality adds QC value: Combining RP and HILIC provided complementary selectivity—HILIC separated polar sequence variants and deamidation species that were unresolved in RP, while RP provides established retention behavior for hydrophobic peptides. Mass spectral data were crucial to detect co‑eluting impurities that UV detection missed.

Benefits and practical applications

• Enhanced impurity detection: Orthogonal LC modes plus MS improve confidence in raw material screening and batch release testing by revealing impurities invisible to single‑mode UV methods.
• Method robustness and QC fit: HILIC screening can be incorporated into QC workflows to identify problematic impurities early; RP methods remain useful for routine assays but should be complemented by orthogonal checks.
• Guidance for method development: Systematic tuning of pH, ionic strength and ion‑pairing reagents offers a practical path to resolve closely related peptide variants without extreme column temperature manipulation.

Future trends and potential applications

• Routine incorporation of orthogonal LC (RP + HILIC) with MS detection in QC and stability testing of peptide therapeutics, particularly as GLP‑1 analogs diversify and market pressure increases.
• Greater use of high‑resolution MS, sequence confirmation workflows and software integration (e.g., UNIFI) for automated impurity identification and quantitation.
• Adoption of multi‑dimensional LC or LC×MS approaches, ion mobility separation and advanced data processing to further disentangle complex impurity profiles.
• Application of these workflows across peptide classes (multiagonists, modified analogs) to meet evolving regulatory expectations on impurity characterization.

Conclusions

Orthogonal LC‑UV/MS workflows combining RP and HILIC separations substantially improve impurity profiling for GLP‑1 analogs such as exenatide. HILIC chemistries (BEH HILIC and BEH Amide) effectively resolve sequence insertion and deamidation species that can coelute under RP, while MS detection is essential to detect and confirm mass variants. Optimizing mobile phase pH, ionic strength and counter‑ions enables tuning of retention and elution order without resorting to non‑robust conditions. These approaches strengthen QC screening, raw material assessment and stability testing for peptide therapeutics.

Reference

1. Han D, Birdsall R, Bhiwankar N. Accelerating Method Development and Manufacturing of GLP‑1 Analogs with LC‑UV/MS. Waters Corporation Application Note 720008800, May 2025.