Application of LC-UV/MS Workflows to Increase Efficiency in Impurity Profiling of GLP-1 Analogs

Importance of the topic

Glucagon-like peptide-1 receptor agonists play a crucial role in managing type 2 diabetes and obesity. Efficient impurity profiling of GLP-1 analogs is essential for ensuring drug safety, accelerating method development and manufacturing scale-up, and meeting stringent regulatory requirements.

Study objectives and overview

This study demonstrates the integration of liquid chromatography with UV and mass detection to enhance impurity profiling workflows for GLP-1 analogs. Key aims include rapid identification of stress-induced degradants under oxidation, pH, and thermal stress and support for raw material screening, process control, lot release, and stability monitoring.

Methodology and Instrumentation

Samples: Research-grade exenatide and tirzepatide stocks formulated in acetate or phosphate buffers
Forced degradation: Oxidative (hydrogen peroxide), thermal (50 °C, 30 days), enzymatic digestion (RapiZyme Trypsin)
Chromatography: Arc Premier UPLC system with XSelect Premier Peptide CSH C18 column (2.5 µm, 4.6×100 mm), gradient elution with 0.1% formic acid in water/acetonitrile, flow rate 0.96 mL/min, column temperature 60 °C, sample temperature 10 °C, injection 10 µL
Detection: ACQUITY QDa II Mass Detector (ESI+ full scan 250–1500 m/z, scan rate 5 Hz), UV at 220 nm, Empower CDS for data acquisition and reporting

Main Results and Discussion

Thermal stress of exenatide produced seven new peaks in the UV chromatogram. Mass data allowed putative assignment of a nonenzymatic Asp–Leu cleavage (3,254.1 Da) and pinpointed a thermally labile site.
Oxidative stress of exenatide revealed a +16 Da mass shift. Trypsin digestion and LC-UV/MS identified oxidized T2 peptide fragment, implicating methionine as a primary oxidation site.
Tirzepatide thermal stress generated low-abundance peaks. MS data confirmed the fatty acid conjugate fragment (342.5 m/z) and peptide moiety (1123.5 m/z), providing specificity beyond UV detection.

Benefits and Practical Applications

• Compact, inline mass detection lowers barriers to MS adoption in QA/QC labs.
• Orthogonal mass data accelerates impurity identification and risk-based decision-making.
• Enhanced confidence in stability-indicating methods supports regulatory compliance and product safety.
• Streamlined workflows improve laboratory productivity and reduce errors.

Future trends and potential applications

Integration of compact mass detectors with automated platforms and AI-driven data analysis will further accelerate biopharmaceutical characterization. Expansion of LC-UV/MS workflows to other peptide and protein therapeutics, high-throughput impurity screening, and real-time process monitoring are anticipated developments.

Conclusion

Inline LC-UV/MS workflows enabled by the ACQUITY QDa II Mass Detector provide a robust, user-friendly approach for impurity profiling of GLP-1 analogs. Access to orthogonal mass information expedites method development, enhances product understanding, and supports regulatory and manufacturing needs.

References

1. Yu M et al. Battle of GLP-1 Delivery Technologies. Advanced drug delivery reviews. 2018;130:113-130. DOI: 10.1016/j.addr.2018.07.009.
2. Birdsall RE, Du X, Nyholm K. Extending the Analytics of Biopharmaceutical QA/QC Labs with the ACQUITY QDa II Mass Detector. Waters Application Note. June 2024.
3. Han D, Ippoliti S, Birdsall RE, Nyholm K. Accelerating Method Development and Manufacturing of GLP-1 Analogs with LC-UV/MS. Waters Application Note. May 2025.