High Mass Loading of Peptides with Hybrid Particle C18 Columns and Acetic Acid Mobile Phases

Importance of the Topic

Peptide purification by preparative reversed-phase chromatography is critical for producing high-purity therapeutic agents and analytical markers. Minimizing co-elution of closely related impurities ensures reliability in biological assays and safety in pharmaceutical products. Reducing reliance on toxic ion-pairing agents like trifluoroacetic acid (TFA) by using acetic acid (HOAc) mobile phases can streamline workflows and yield pharmaceutically acceptable peptide salts in fewer purification steps.

Objectives and Study Overview

This study evaluates two hybrid-particle C18 column chemistries (BEH130 C18 and CSH130 C18) under high mass load conditions. Key goals include:

• Comparing resolution and peak shape with mobile phases modified by 0.1% TFA versus optimal HOAc concentrations.
• Assessing loadability for a nine-component peptide mixture and a low-purity synthetic peptide.
• Demonstrating the potential to simplify purification by avoiding TFA and using HOAc phases.

Methodology and Instrumentation

Analytical and semi-preparative separations were performed on an ACQUITY UPLC H-Class Bio System coupled to a Xevo G2 Q-Tof mass spectrometer. Key details include:

• Columns: XBridge BEH130 C18 and XSelect CSH130 C18 (4.6×100 mm, 5 µm, 130 Å).
• Mobile phases: 0.1% TFA or varying HOAc concentrations (0.1% and 1%) in water and 90:10 ACN/water.
• Sample loads: 30–500 µg of a nine-peptide mixture and 50 µg–4 mg of synthetic peptide DFVGYGVKDFVGVGVK.
• Detection: UV at 214 nm and 250 nm; MS detection by ESI+ over 50–1990 m/z.

Main Results and Discussion

Loading studies with the nine-component peptide mixture revealed that CSH130 C18 maintained narrow peak widths under both 0.1% TFA and 0.1% HOAc, while BEH130 C18 showed peak broadening with HOAc. Average 4σ peak widths for semi-preparative loads were 0.5–0.6 min on CSH130 C18 and increased from 0.8 min (TFA) to 1.5 min (HOAc) on BEH130 C18.

For the low-purity synthetic peptide, HOAc concentration strongly influenced peak shape at preparative loads (1 mg). BEH130 C18 yielded optimal peaks with 0.1% HOAc, whereas CSH130 C18 required 1% HOAc to prevent fronting. Both columns with optimized HOAc phases delivered narrower peaks (0.5–0.6 min) compared to 0.1% TFA (0.6–1.1 min).

High-load injections up to 4 mg demonstrated stable and predictable peak profiles, especially on CSH130 C18, indicating suitability for large-scale preparative applications.

Practical Benefits and Applications

Use of HOAc mobile phases with hybrid C18 columns offers:

• Streamlined purification workflows by avoiding toxic TFA removal steps.
• Improved chromatographic resolution and narrower fractions for higher purity.
• Enhanced loadability for increased throughput in peptide manufacturing.
• Complementary column chemistries (BEH and CSH) to tackle challenging separations.

Future Trends and Opportunities

Emerging directions include:

• Fine-tuning HOAc concentrations for additional peptide classes and complex mixtures.
• Scaling methods to preparative columns with larger diameters for industrial production.
• Integrating charged-surface chemistries in continuous purification platforms.
• Exploring alternative benign mobile phase additives to further reduce environmental impact.

Conclusion

Hybrid-particle BEH130 C18 and CSH130 C18 columns effectively support high mass loading peptide separations with acetic acid mobile phases, delivering comparable or superior peak sharpness to TFA-containing methods. Optimizing HOAc concentration for each stationary phase enables streamlined purifications, improved resolution, and enhanced productivity in peptide manufacturing.

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