Optimization of RPLC-UV methods for impurity analysis of the GLP-1 receptor agonists semaglutide and tirzepatide

Optimization of RPLC-UV methods for impurity analysis of semaglutide and tirzepatide

Importance of the topic

• GLP-1 receptor agonists such as semaglutide and tirzepatide are high-value, fatty-acid–modified peptide therapeutics with rapidly expanding clinical and commercial use. Robust impurity control is essential for safety, efficacy, and regulatory submissions because peptide-related impurities are often structurally very similar to the API and can arise during synthesis, formulation, storage, and transport.
• Reversed-phase liquid chromatography (RPLC) remains a primary tool for peptide impurity profiling; however, method development is challenging due to subtle sequence changes and lipid modifications that affect peptide hydrophobicity and conformation. Practical guidance and ready-to-use conditions help streamline impurity method development for GLP-1 RAs.

Objectives and study overview

• Systematically evaluate key chromatographic variables (stationary phase chemistry, organic solvent, acidic modifier, column temperature, and gradient shape) to define optimized RPLC-UV methods for impurity analysis of semaglutide and tirzepatide.
• Compare Hypersil GOLD C4 and Hypersil GOLD Peptide stationary phases, MeOH vs ACN organic phases, and formic acid (FA) vs trifluoroacetic acid (TFA) additives using thermally stressed samples to generate representative impurity profiles.
• Deliver practical, optimized RPLC-UV methods suitable for impurity separation, with commentary on method trade-offs (chromatographic performance vs LC–MS compatibility).

Methodology

• Sample preparation: APIs (purity >99%) were dissolved at 1 mg/mL in 20% ACN/water; thermal stress was applied at 60 °C with shaking (50 rpm) for 7 days to induce impurities. Injected amount: 5 µL per run (≈5 µg API).
• Screening design: Fixed, solvent-specific gradients were used to compare combinations of two columns (Hypersil GOLD C4, Hypersil GOLD Peptide) with four mobile phase permutations (ACN or MeOH combined with 0.1% FA or 0.1% TFA). Column temperatures of 30, 40, 50 and 60 °C were evaluated. Performance ranking used count of impurity peaks (>0.1% relative area) and consideration of API peak shape and resolution to adjacent impurities.
• After screening, gradient shape (shallower slopes, extended windows) and column temperature were optimized for the top-ranking column/mobile-phase pairs to maximize impurity resolution and uncover coeluted species.

Used instrumentation

• Thermo Scientific Vanquish Flex UHPLC System (Vanquish System Base, Binary Pump F, Split Sampler FT, Column Compartment H, Diode Array Detector with Standard Bio Flow cell).
• Columns: Hypersil GOLD C4, 150 × 2.1 mm, 1.9 µm; Hypersil GOLD Peptide, 150 × 2.1 mm, 1.9 µm.
• Chromatography Data System: Thermo Scientific Chromeleon CDS 7.3.2. UV detection at 280 nm; autosampler at 5 °C; flow 0.3 mL/min.

Main results and discussion

• Mobile phase composition (organic solvent and acidic additive) was the dominant factor controlling peptide selectivity; column ligand chemistry (C4 vs peptide) had a smaller, but still relevant, effect.
• TFA-containing mobile phases produced superior peak shape and resolution in many tested combinations, while FA-containing systems—though often giving slightly inferior chromatographic appearance—were retained because of LC–MS compatibility for follow-up analyses.
• Solvent-specific temperature behavior: with MeOH-based mobile phases, retention times decreased markedly as temperature increased; conversely, with ACN-based mobile phases, retention times increased slightly with temperature. These trends reflect solvent–peptide interaction differences and possible temperature-driven peptide conformational changes, particularly relevant for fatty-acid–modified peptides.
• Column temperature typically improved impurity resolution; e.g., raising temperature to 60 °C resolved impurities that coeluted at lower temperatures for tirzepatide. However, this was not universal: in a few combinations (notably semaglutide on C4 with TFA), raising temperature reduced resolution and induced coelution, highlighting the need to tailor temperature to the specific column/mobile-phase/analyte combination.
• Top-ranked screening conditions for both peptides included Hypersil GOLD C4 with MeOH–TFA for early stages; after targeted gradient optimization (shallower gradients and extended time windows), additional impurities became visible and total relative impurity area increased by ~1.5% for semaglutide and ~2.1% for tirzepatide (TFA-based methods), demonstrating that gradient refinement can uncover coeluted species.
• Tirzepatide generally showed longer retention than semaglutide under identical conditions, indicating that small sequence or lipidation differences substantially influence chromatographic behavior and necessitate analyte-specific optimization rather than a one-size-fits-all platform.

Benefits and practical applications of the methods

• Provides ready-to-use, systematically derived RPLC-UV conditions that laboratories can adopt or adapt for routine impurity profiling of semaglutide and tirzepatide during development, manufacturing, and stability testing.
• Guides method developers through a stepwise approach: (1) screen stationary phases and mobile-phase chemistries, (2) evaluate column temperature effects, and (3) optimize gradient shape to maximize impurity detection and resolution.
• Offers practical trade-offs: use TFA-containing methods for best chromatographic performance when UV-only workflows are primary; preserve FA-based options when subsequent LC–MS characterization is needed for impurity identification.

Future trends and possible applications

• Greater integration of LC–MS workflows with optimized RPLC separations to expedite structure elucidation of low-level peptide impurities, leveraging MS-friendly mobile phases or post-column approaches to retain chromatographic quality while enabling identification.
• Method transfer and robustness studies to support regulatory submissions (e.g., ANDAs) will drive standardized screening strategies and platform elements for lipidated peptides, but analyte-specific tailoring will remain essential.
• Emerging approaches—multidimensional chromatography, stationary phases designed for lipidated/helical peptides, higher-temperature UHPLC operation, and computational modeling (QbD) of peptide retention/selectivity—will accelerate development of stability-indicating methods and improve impurity coverage.
• Application of high-resolution MS, automated peak deconvolution, and machine-learning tools to predict likely impurity species and guide targeted method refinements.

Conclusion

• A structured, empirical RPLC development workflow combining column/mobile-phase screening, temperature evaluation, and gradient optimization produced robust UV-based impurity analysis methods for semaglutide and tirzepatide. The study highlights that mobile phase composition and temperature are key levers for selectivity; TFA improves chromatographic performance while FA preserves LC–MS compatibility. Because small structural differences between GLP-1 RAs substantially affect retention and selectivity, method development must be compound-specific and include targeted gradient refinement to reveal coeluted impurities.

References

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4. Gilar M., Jaworski A., McDonald T. S. Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides. Journal of Chromatography A, 2014, 1337, 140.