Separation of a GLP-1 Receptor Agonist and Structurally Similar Impurities Using BioResolve™ Peptide Phenyl‑Hexyl+ and C18 + Columns

Significance of the topic

Peptide therapeutics such as GLP-1 receptor agonists are rapidly expanding in biopharmaceutical development. High-resolution chromatographic methods are essential for separating and characterizing structurally similar peptide variants (deamidation products, isoaspartate, single-residue D-enantiomers, and single amino-acid substitutions) that can affect product safety, stability, and efficacy. Robust, selective reversed-phase (RP) separations support impurity profiling, release testing, and method development for regulatory and manufacturing needs.

Objectives and study overview

The presented application brief evaluates the performance and complementary selectivity of two Waters BioResolve peptide RP stationary phases—Phenyl‑Hexyl+ and C18+—for separation of tirzepatide and seven known spiked impurities. Key aims were to demonstrate: whether D‑amino acid impurities can be resolved from the main peptide peak; how deamidation isomer species behave on different chemistries; and the effect of column length on resolution under comparable run times.

Methodology

The study used superficially porous particle peptide columns (1.6 µm particle size, 230 Å pore size) with positive surface charge modification and MaxPeak Premier inert hardware. Separations were run with formic-acid–modified mobile phases and shallow gradients to emphasize selectivity differences between stationary phases. Comparative experiments included 2.1 x 150 mm and 2.1 x 300 mm column lengths to assess the impact of column length on resolution.

Instrumentation used

• Columns: BioResolve Peptide Phenyl‑Hexyl+ and BioResolve Peptide C18+, superficially porous particles (~1.6 µm, 230 Å).
• Column dimensions tested: 2.1 x 150 mm and 2.1 x 300 mm.
• Mobile phases: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile.
• Gradients: shallow gradients over long windows (examples: ~32–36% B in 90 min and ~35–39% B in 30 min for 150 mm; comparable but shifted windows for 300 mm).
• Flow rate: 0.3 mL/min.
• Column temperature: 50 °C.
• Detection: UV at 214 nm.
• System pressures reported (excluding system contribution): ~6,320 psi for 150 mm; ~12,150 psi for 300 mm.

Main results and discussion

Key experimental observations and interpretations include:

• Both Phenyl‑Hexyl+ and C18+ columns separated tirzepatide from a D‑Ser32 impurity (Impurity 7), demonstrating capability to resolve certain D‑amino‑acid isomers for large peptides.
• Deamidation isomers (Impurities 1–3) exhibited different elution orders on the two chemistries: Phenyl‑Hexyl+ separated Impurity 3 from 1 and 2, while C18+ separated Impurity 2 from 1 and 3. This highlights the complementary selectivity of aromatic/π-interaction–tuned versus alkyl C18 phases.
• Impurity 6 (Ile → Norvaline) coeluted or was not fully resolved from the main peak on the 150 mm columns using shallow gradients, indicating limits of resolution for very closely related substitutions under those conditions.
• Extending column length to 300 mm improved resolution substantially for several critical pairs (including partial separation of Impurity 1 and 2 and better separation of species flanking the main peak), even when run time and nominal gradient windows were held comparable. This suggests column length provided greater incremental peak capacity than simply extending gradient time on the shorter column in these cases.
• Surface modifications—controlled positive charge on the particle surface—and MaxPeak Premier inert hardware contributed to improved peak shape, loading capacity for basic peptides, and reduced non-specific adsorption, supporting robust performance with formic-acid mobile phases.

Benefits and practical applications

• Complementary stationary phases: Using both Phenyl‑Hexyl+ and C18+ provides orthogonal selectivity useful during method development to resolve closely related peptide variants.
• Improved analysis of critical impurities: Ability to separate D‑amino‑acid isomers and some deamidation species supports impurity identification and quantitation for GLP‑1 therapeutics.
• Scalability of resolution: Selecting a longer column (300 mm) delivers higher resolution for challenging separations without increasing run time, albeit with higher backpressure requirements.
• Robustness for basic peptides: Controlled surface charge and inert hardware minimize peak tailing and nonspecific losses when using acidic mobile phases, improving method ruggedness and recovery.

Future trends and applications

• Broader adoption of superficially porous particle chemistries with tailored surface charges to balance retention, peak shape, and loading for large peptides and peptide‑based biologics.
• Integration of orthogonal RP chemistries early in impurity profiling to accelerate identification of isomeric and post‑translational variants.
• Continued optimization of column dimensions and gradient strategies to trade off throughput versus resolution—longer narrow-bore columns will remain a practical option for highest-resolution impurity analysis where instrument pressure limits permit.
• Combined use with high‑resolution mass spectrometry for peak identification after chromatographic separation, particularly for species that coelute on a single chemistry.

Conclusion

BioResolve Peptide Phenyl‑Hexyl+ and C18+ columns effectively separate tirzepatide from several structurally similar impurities, with each chemistry offering distinct selectivity that can be exploited during method development. While some variants require longer columns or shallower gradients for baseline separation, surface charge modification and inert hardware enhance peak shape and robustness for peptide analyses. Selecting complementary stationary phases and appropriate column length are practical strategies for improving impurity resolution in GLP‑1 peptide workflows.

References

1. Lauber M.A.; Koza S.M.; Fountain K.J. Increasing Peak Capacity in Reversed-Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns. Waters Application Note. 720004568, 2013.
2. Improving Chromatographic Separations of Biopharmaceuticals with MaxPeak High Performance Surfaces (HPS) Technology. Waters eBook. 720008110, 2024.
3. Yang H.; Warren B.; Koza S. Development of Monoclonal Antibody Charge Variant Analysis Methods Using a BioResolve SCX mAb Column. Waters Application Note. 720006477, 2019.