Advanced 2D-LC/MS Workflow for the Characterization of Semaglutide and Its Impurities

Význam tématu

Peptide-based therapeutics such as semaglutide play a pivotal role in modern medicine, offering high target specificity and favorable pharmacokinetics. Accurate separation and identification of semaglutide-related impurities are critical for ensuring drug safety, efficacy, and compliance with stringent regulatory standards. Advanced analytical workflows combining multidimensional chromatography and high-resolution mass spectrometry address challenges arising from closely related peptide variants and MS-incompatible buffers.

Cíle a přehled studie / článku

This work demonstrates an online two-dimensional liquid chromatography (2D-LC) workflow coupled to time-of-flight mass spectrometry (Q-TOF) for comprehensive characterization of semaglutide and its by-products. The objectives include:

• Achieve efficient separation of semaglutide impurities coeluting in conventional one-dimensional methods.
• Enable impurity desalting and MS compatibility via a heart-cutting 2D-LC approach.
• Apply accurate mass detection and MS/MS fragmentation for sequence confirmation of synthetic and truncated peptide variants.

Použitá metodika a instrumentace

The analytical scheme employs:

• First dimension: Agilent 1290 Infinity III bio 2D-LC equipped with an AdvanceBio Peptide Plus C18 column and phosphate buffer-based mobile phase for high chromatographic resolution.
• Second dimension: Agilent 1290 Infinity III bio 2D-LC with AdvanceBio RP-mAb C4 column and volatile formic acid–acetonitrile gradients for desalting and MS compatibility.
• Detection: Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI source operated in extended dynamic range for accurate mass and high-sensitivity MS/MS data.
• Sampling: Multiple heart-cutting (MHC) loops and high-resolution (HiRes) multi-injection to capture narrow impurity peaks across the chromatogram.
• Software: Agilent MassHunter BioConfirm 12.1 for automated intact mass matching and sequence confirmation of semaglutide and its variants.

Hlavní výsledky a diskuse

Implementation of the 2D-LC/Q-TOF workflow enabled:

• Baseline separation of low-abundance peptide impurities that coelute with the main semaglutide peak in 1D LC.
• Identification of truncated sequences (e.g., missing histidine or α-methylalanine) and isomeric forms via exact mass within ±5 ppm and diagnostic MS/MS fragmentation patterns.
• Efficient desalting step in the second dimension preserving first-dimension resolution, yielding high-quality spectra for both intact peptide and truncation products.
• Throughput gains achieved by HiRes multi-injection, allowing up to ten heart-cut fractions per run without hardware modifications.

Přínosy a praktické využití metody

The described 2D-LC/Q-TOF strategy offers:

• Robust impurity profiling essential for quality control in peptide drug development and manufacturing.
• Enhanced confidence in sequence confirmation and identification of post-synthesis modifications.
• Improved regulatory compliance by comprehensive detection of potential degradation products.
• Adaptability to other peptide or protein therapeutics requiring MS-incompatible buffers in primary separation.

Budoucí trendy a možnosti využití

Future directions include:

• Integration of real-time data analytics and machine learning for automated peak selection in multidimensional workflows.
• Expansion to 3D-LC techniques for even greater resolution of highly complex peptide mixtures.
• Application to biosimilars and advanced bioconjugates where minor structural variants demand high-precision analysis.
• Miniaturization of 2D-LC interfaces and microfluidic platforms to reduce solvent consumption and increase throughput.

Závěr

The advanced 2D-LC/Q-TOF workflow combines orthogonal chromatographic dimensions with high-resolution mass spectrometry and specialized software to deliver comprehensive impurity characterization of semaglutide. This methodology enhances separation power, ensures MS compatibility, and provides reliable identification of peptide variants, supporting rigorous quality control in biopharmaceutical research and production.

Reference

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