Confirmation of Peptide-Related Impurity Intact Mass Using Agilent 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT

Significance of the Topic

Peptide-related impurities pose critical challenges in biopharmaceutical development and quality control due to their structural diversity, high molecular weights and potential immunogenicity. Regulatory guidelines require strict monitoring of impurities above 0.5 % to ensure patient safety and product efficacy. Effective analytical strategies are essential for generic drug manufacturers and stability studies to detect, separate and confirm the molecular identities of peptide impurities.

Objectives and Study Overview

This study aimed to demonstrate a two-dimensional liquid chromatography (2D-LC) approach coupled with mass spectrometry (MS) to characterize forced-degradation impurities of Semaglutide. Thermal and acidic stress conditions were applied to generate degradation products, which were then separated using Agilent® 1290 Infinity II Bio 2D-LC and identified by Agilent InfinityLab LC/MSD XT.

Methodology and Instrumentation

The workflow combined high-resolution reversed-phase separation in two dimensions with MS-compatible mobile phases and software deconvolution.

• First Dimension (1D): AdvanceBio Peptide Plus column under 0.4 % TFA at 60 °C, 0.4 mL/min, using a shallow gradient to resolve closely eluting impurities.
• Heart-Cutting: Time-based multiple heart-cut (MHC) valve transferred selected peaks into the second dimension via 40 µL loops.
• Second Dimension (2D): Poroshell 120 CS-C18 column with 0.1 % formic acid in water/acetonitrile, 0.6 mL/min, 13 min cycle including equilibration.
• Detection: LC/MSD XT with positive-mode ESI, gas temperature 325 °C, scan range m/z 500–2,500, and OpenLab CDS v2.8 deconvolution for intact mass determination.

Main Results and Discussion

Under thermal stress (60 °C, 2 days), six characteristic impurity peaks were resolved and heart-cut into 2D. Total ion chromatograms (TICs) showed improved separation of coeluting species. Deconvoluted masses ranged from 3,152 Da to 4,160 Da, revealing truncations and modifications relative to native Semaglutide (4,113.6 Da).
Under acidic stress (0.1 M HCl, 60 °C, 2 days), eleven degradation products were identified and analyzed. Deconvolution confirmed masses between 2,582 Da and 4,114 Da, indicating varied sequence fragments and chemical alterations. Low-level impurities yielded interpretable MS spectra despite low UV signals.

Benefits and Practical Applications

This 2D-LC/MS approach enables robust characterization of peptide degradants in formulation development, forced-degradation testing and generic drug approval. Key advantages include:

• Enhanced separation of structurally similar impurities under MS-friendly conditions.
• Accurate intact mass confirmation via software deconvolution, supporting impurity identification and quantitation.
• Compatibility with biocompatible materials and high-throughput workflows for QA/QC laboratories.

Future Trends and Opportunities

Emerging prospects include integrating high-resolution Q-TOF MS for sequence confirmation, automating heart-cut selection via intelligent software control, and applying the 2D-LC/MS strategy to other complex peptides and biotherapeutics. Advanced data-processing algorithms may further streamline impurity profiling and regulatory reporting.

Conclusion

The combination of Agilent 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT provides a powerful platform for separating, detecting and confirming peptide-related impurities. The workflow meets MS-compatibility requirements while delivering high sensitivity and reliable intact mass assignments, aiding peptide drug developers in maintaining product quality and compliance.

Used Instrumentation

• Agilent 1290 Infinity II Bio 2D-LC: flexible pump, high-speed pump, multisampler, multicolumn thermostat, diode array detectors, valve drives with MHC and ASM configurations.
• Agilent InfinityLab LC/MSD XT for ESI(+)-MS detection.
• Agilent OpenLab CDS software version 2.8 for deconvolution.

References

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