GLP-1 Analog: Accelerating Method Development and Manufacturing with LC-UV/MS

Significance of the topic

Glucagon-like peptide-1 (GLP-1) analogs have become critical therapeutic agents for type 2 diabetes and weight management. Their peptide structure and fatty acid conjugation improve half-life and potency but add complexity to quality control. Advanced analytical workflows that combine liquid chromatography with ultraviolet and mass spectrometric detection (LC-UV/MS) are essential to characterize purity, degradation pathways, and impurities that might be missed by UV detection alone.

Objectives and study overview

This study aimed to accelerate method development and streamline manufacturing control for two GLP-1 analogs (exenatide and tirzepatide) by integrating an LC-UV/MS workflow. Key goals included rapid impurity screening, detailed degradation profiling under thermal and oxidative stress, and seamless incorporation of mass detection into existing QC platforms.

Methodology and instrumentation

Samples of exenatide and tirzepatide were prepared in their formulated drug products and analyzed by LC-UV and MS. Main instrumentation and conditions included:

• Empower Chromatography Data System 3.8.1 for data acquisition and processing
• ACQUITY Arc Premier System coupled with ACQUITY QDa II Mass Detector for LC-UV/MS analysis
• XSelect Premier Peptide CSH C18 Column (130 Å, 2.5 µm, 4.6 × 100 mm) at 60 °C
• Mobile phases: water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B)
• Gradient elution (1%–95% B) at 0.96 mL/min
• PDA detection from 210–400 nm and MS positive ion scan (250–1500 Da, centroid)

Main results and discussion

The combined LC-UV/MS workflow demonstrated several advantages:

• Orthogonal mass screening unveiled a +97.1 Da impurity in one exenatide vendor sample that was undetected by UV alone.
• Oxidative stress (3% H₂O₂, 24 h) on exenatide produced four distinct oxidation products; MS spectra located the susceptible methionine and tryptophan residues.
• Tirzepatide thermal stress revealed cleavage of its fatty acid linker. Extracted ion chromatograms (XIC) of the free fatty acid (342.5 m/z) and conjugate fragment (1123.5 m/z) gave insight into degradation pathways.
• The ACQUITY QDa II detector integrated easily onto existing LC-UV platforms, delivering sensitive mass confirmation without extensive MS expertise.

Benefits and practical applications

The integrated LC-UV/MS approach offers:

• Enhanced impurity profiling and degradant identification for regulatory compliance.
• Streamlined raw material screening and lot release testing with orthogonal confirmation.
• Improved stability monitoring under stress conditions to inform formulation strategies.
• Greater confidence in method robustness and data integrity during development and manufacturing.

Future trends and applications

Emerging directions in GLP-1 analog analysis include:

• Adoption of high-resolution and multi-mode MS detectors for deeper structural elucidation.
• Automated and real-time in-line monitoring using AI-driven data analysis.
• Microflow and ultrahigh-performance LC integrating MS for faster separations and reduced solvent use.
• Broader deployment of LC-UV/MS in regulated QC laboratories to accelerate product approvals.

Conclusions

The study validated that integrating the ACQUITY QDa II Mass Detector into routine LC-UV workflows significantly enhances impurity detection and degradant characterization of GLP-1 analogs. Orthogonal mass data improves decision making and supports accelerated method development, manufacturing control, and regulatory compliance.

References

1. D. Han et al. Accelerating Method Development and Manufacturing of GLP-1 Analogs with LC-UV/MS. Application Note 720008800, May 2025, Waters Corporation.
2. D. Han et al. Application of LC-UV/MS Workflows to Increase Efficiency in Impurity Profiling of Peptide Therapeutics, 2025, Waters Corporation.