Analysis of a Synthetic Peptide and Its Impurities

Significance of the topic

The reliable separation and characterization of synthetic peptides and their impurities is critical for quality control in biopharmaceutical development. Peptide therapeutics, such as bivalirudin, require precise analytical methods to detect sequence variants, degradation products, and modifications that can affect efficacy and safety.

Objectives and overview of the study

This application note evaluates an LC method using an Agilent AdvanceBio Peptide Plus column with formic acid (FA) as a mass spectrometry (MS)–compatible modifier. The goals were to achieve high-resolution peptide separations, enable seamless transfer between LC/UV and LC/MS workflows, and identify major impurities in an aged bivalirudin sample.

Methodology and instrumentation

The chromatographic method employed a 2.1×150 mm AdvanceBio Peptide Plus column at 60 °C with a gradient from 17 % to 95 % acetonitrile in 0.1 % FA. Flow rate was 0.4 mL/min. UV detection used a 5 µL injection, while MS detection used a 1 µL injection.

Used Instrumentation

• Agilent 1290 Infinity II binary pump, autosampler, and column compartment
• Agilent 1260 Infinity II diode array detector for LC/UV
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent Jet Stream source for LC/MS and MS/MS
• Data processed with Agilent OpenLab 2.2 CDS (UV) and MassHunter BioConfirm B.08.00 (MS/MS)

Main results and discussion

The LC/UV chromatogram of aged bivalirudin revealed five major peaks. High-resolution MS and MS/MS identified:

• Peak 1 (m/z 2,049.9467; –129 Da): deletion of a glutamic acid residue confirmed by b15 and y4 fragments
• Peak 2 (m/z 2,178.9894): full-length bivalirudin (monoisotopic mass 2,178.9858 Da)
• Peak 3 (m/z 2,121.9663; –57 Da): glycine deletion
• Peak 4 (m/z 2,160.9764; –18 Da): dehydration (loss of H₂O)
• Peak 5 (m/z 2,179.9742; +1 Da): Asn deamidation to Asp verified by y5 and b15 MS/MS ions

The AdvanceBio Peptide Plus stationary phase, bearing a positively charged hybrid surface, provided sharp peaks and high resolution with FA, overcoming the tailing issues found on traditional C18 columns.

Benefits and practical applications

This method delivers:

• Enhanced peptide separation using MS-friendly mobile phases
• Direct compatibility with both UV and MS detection modes
• Robust identification of sequence variants and degradation products
• Streamlined transfer between QC LC/UV assays and LC/MS impurity profiling

Future trends and possibilities

Advancements in peptide chromatography may include further optimization of surface chemistries to improve peak capacity with volatile modifiers. Integration with automated sample handling and advanced MS/MS data analysis will support high-throughput impurity screening. Emerging software tools for de novo sequencing and impurity quantitation are expected to enhance method sensitivity and specificity.

Conclusion

The combination of an Agilent AdvanceBio Peptide Plus column with formic acid mobile phases achieves high-resolution separation and confident MS/MS identification of synthetic peptide impurities. The approach unifies LC/UV and LC/MS workflows for efficient quality control of peptide therapeutics.

References

1. Eggen I. et al. Control Strategies for Synthetic Therapeutic Peptide APIs Part III: Manufacturing Process Considerations. Pharm. Technol. 2014, 38(5).