Altura Size Exclusion 130 Å Allows Comprehensive Analysis of GLP-1 Analogues Over a Broad pH Range

Altura Size Exclusion 130 Å Enables Comprehensive Analysis of GLP-1 Analogues Across a Broad pH Range

Importance of the topic

Glucagon-like peptide-1 receptor agonists (GLP-1s) are an expanding therapeutic class with numerous closely related impurities and frequent chemical modifications (notably fatty-acid conjugation at Lys26) that complicate chromatographic analysis. Accurate size-exclusion chromatography (SEC) methods are critical for monitoring aggregation, fragment content and overall product quality — especially as many GLP-1 patents expire and biosimilar development accelerates. Minimizing non-specific adsorption to silica and metal surfaces is essential to obtain symmetric peaks, preserve aggregate information and ensure reproducible quantitation.
Study objectives and overview

This study evaluated how pH and organic solvent fraction (acetonitrile, ACN) influence aggregation behavior and peak shape of three GLP-1 analogues (liraglutide, semaglutide, exenatide). It compared performance of Agilent AdvanceBio SEC material packed into standard stainless-steel hardware, Agilent Altura Ultra Inert hardware (Altura SEC 130 Å and 300 Å), and a competitor inert SEC column. The goal was to define method-development space (pH, ACN, pore size) and quantify gains obtained by Ultra Inert hardware for SEC analysis of GLP-1 analogues.
Methodology and instrumentation

Instrumentation and software (used in the study):

• Agilent Infinity III 1260 multiple wavelength detector (G7165A)
• Agilent Infinity III 1260 multicolumn thermostat (G7116A)
• Agilent Infinity III 1260 Bio multisampler (G5668A)
• Agilent Infinity III 1260 Bio-Inert pump (G5654A)
• Agilent OpenLab CDS Acquisition and Analysis v2.8

Key chromatographic parameters (summary):

• Columns evaluated: AdvanceBio SEC 130 Å (4.6 × 300 mm, 2.7 µm), Altura SEC 130 Å (Ultra Inert), Altura SEC 300 Å (Ultra Inert), competitor inert SEC (125 Å, 4.6 × 300 mm, 2.5 µm)
• Flow rate: 0.35 mL/min; temperature: 25 °C; injection: 1 µL; run time: 20 min
• Detection: UV 220 nm; data rate 10 Hz
• Mobile phases: Eluent A = 1× PBS pH 7.4; B = 25:65 acetic acid:1 g/L arginine, pH 2; C = 0.1% ammonium trifluoroacetate, pH 8.5; D = acetonitrile (ACN)
• Nine test conditions mixing A:B:C:D in ranges from mostly PBS to high ACN and low/high pH (see study for full matrix)

Sample and reagent notes:

• Samples prepared at 1 mg/mL in 1× PBS pH 7.4
• High-purity water and HPLC-grade reagents; peptides sourced from commercial suppliers

Main results and discussion
Key experimental findings:

• pH strongly affects aggregation and elution behavior: increasing pH from 2 to 8.5 produced a decrease in elution volume for all three GLP-1s, consistent with formation of higher-order oligomers (literature reports aggregation around pH 6–9).
• Organic content (ACN) is critical for peak shape of lipidated GLP-1s: liraglutide and semaglutide (both modified with fatty-acid chains at K26) exhibited markedly improved peak symmetry with increased ACN. Low ACN (e.g., 10%) caused severe broadening and carryover for liraglutide; semaglutide also showed very wide peaks at low ACN. Exenatide (no fatty-acid conjugate) was far less sensitive to ACN fraction.
• Pore-size screening is necessary: the wider-pore Altura SEC 300 Å was used to prevent exclusion of higher-order aggregates during initial screening, while the 130 Å column frequently produced sharper main peaks and improved fragment resolution. Thus both pore sizes should be evaluated in method development depending on whether aggregate detection or optimal fragment resolution is prioritized.
• Ultra Inert hardware reduces non-specific adsorption and improves peak symmetry: packing the same SEC stationary phase into Altura Ultra Inert housings significantly reduced peak tailing compared with stainless-steel hardware. Typical reductions in tailing were reported as greater than 60% for liraglutide and exenatide and approximately 30% for semaglutide under most tested conditions; in some comparisons versus a competitor inert column, tailing decreased by up to 75%.
• pH-dependence of metal binding: stainless-steel housings showed increased metal-related adsorption at extreme pH values (pH 2 and 8.5). Altura Ultra Inert hardware allowed more versatile use of both low and high pH conditions with substantially improved reproducibility and peak symmetry, expanding method-design space for modified GLP-1s.
• Effect on aggregate and impurity detection: reduced tailing on Ultra Inert columns preserved peak shapes for aggregates and small fragments. In some cases, stainless-steel columns exhibited tailing that hid or distorted fragment peaks, decreasing resolution and apparent column efficiency for impurity monitoring.

Benefits and practical applications

• Enhanced method robustness: Ultra Inert Altura columns minimize metal interactions and silanol adsorption, improving peak symmetry and reproducibility across a wider pH range without additional method re-optimization.
• Improved impurity and aggregate detection: sharper, less-tailed peaks increase resolution between main species and fragments and preserve aggregate information important for stability and biosimilarity assessments.
• Streamlined development for lipidated peptides: systematic screening of ACN fraction, pH and pore size (130 Å vs 300 Å) is recommended; use of Altura Ultra Inert hardware reduces false negatives caused by adsorption and enables clearer interpretation of aggregation behavior driven by K26 modifications.
• Support for biosimilar development and QC: the improved chromatographic window enables more reliable comparability testing and routine QC for GLP-1 therapeutics and analogues.

Future trends and applications

• Expanded adoption of inert hardware for peptide SEC: as more therapeutic peptides incorporate lipophilic modifications, demand will grow for SEC hardware that mitigates metal and surface interactions.
• Integrated screening workflows: combinatorial screening of pore size, pH and organic fraction will become standard to balance aggregate detectability and fragment resolution for heavily modified peptides.
• Coupling to orthogonal detectors: pairing Ultra Inert SEC with light-scattering or mass-detection approaches will improve characterization of oligomeric states and support advanced biosimilarity studies.
• Tailored stationary phases: further development of polymer-coated or alternative stationary phases optimized for lipidated peptides can reduce residual silanol effects and broaden mobile-phase compatibility.

Conclusion

GLP-1 analogues pose analytical challenges driven by aggregation propensity and hydrophobic modifications at K26. Systematic screening revealed that both pH and ACN fraction markedly influence aggregation, elution volume and peak shape. Agilent Altura Ultra Inert SEC hardware (130 Å and 300 Å) substantially reduces peak tailing attributable to metal binding and surface interactions, enabling a wider and more practical method-design space. Pore-size screening remains important: 300 Å helps reveal larger aggregates while 130 Å can deliver sharper peaks and improved fragment resolution. Overall, Ultra Inert Altura SEC columns improve sensitivity, reproducibility and robustness of SEC analyses for GLP-1 therapeutics, supporting method development and QC for biosimilars and next-generation analogues.
References

1. Giannetti M.; et al. A Spectroscopic and Molecular Dynamics Study on the Aggregation Properties of a Lipopeptide Analogue of Liraglutide, a Therapeutic Peptide Against Diabetes Type 2. Molecules 2023, 28(22), 7536. doi:10.3390/molecules28227536.
2. Castelletto V.; et al. Fibrillar and Micellar Aggregation of Semaglutide and Formation of a Chiral-Imprinted Glass. Biomacromolecules 2026, 27(4), 2818–2827. doi:10.1021/acs.biomac.5c02669.
3. Benet A.; et al. The Effects of pH and Excipients on Exenatide Stability in Solution. Pharmaceutics 2021, 13(8), 1263. doi:10.3390/pharmaceutics13081263.