Streamlined LC-MS Analysis of Stress Induced Impurities of a Synthetic Peptide using the BioAccord System and the waters_connect Intact Mass Application

Significance of the topic

Synthetic peptides are increasingly used in therapeutics due to high biological selectivity, low toxicity, and versatility in treating various diseases.
Impurities generated during peptide synthesis and storage can impact drug safety and efficacy, requiring robust analytical workflows.
Automated, compliance-ready LC-MS methods enable rapid impurity profiling to meet regulatory standards.

Objectives and study overview

This study demonstrates a streamlined LC-MS workflow using the BioAccord system coupled with waters_connect Intact Mass Application for stress-induced impurity analysis of Exenatide, a 39-residue GLP-1 analog.
The approach integrates untargeted and targeted data processing to confirm peptide identity and quantify degradation products under forced conditions.

Methodology

Exenatide samples were prepared in pH 7.5 buffer and incubated at 37 °C and ambient temperature for 2–4 days to induce stress degradation.
Aliquots were diluted in 0.1% formic acid and injected (5 µL) onto an ACQUITY Premier Peptide CSH C18 column with a 15 min LC gradient.
MS data were acquired in positive ESI mode over m/z 50–2000 and deconvoluted using the BayesSpray algorithm (1–15 kDa) for monoisotopic neutral mass determination.

Used instrumentation

• ACQUITY Premier UPLC System with MaxPeak HPS surfaces
• ACQUITY Premier Peptide CSH C18 Column 1.7 µm, 2.1×100 mm
• BioAccord LC-MS System with ACQUITY Premier ion source
• ACQUITY UPLC Tunable UV Detector (214 nm)
• waters_connect v2.0 platform and Intact Mass App v1.2

Key results and discussion

Untargeted deconvolution identified Exenatide’s monoisotopic mass (4184.027 Da) within 5 ppm and revealed five primary impurity peaks.
Targeted analysis assigned modifications such as oxidation, deamidation, methionine loss, succinimide formation, and dimerization based on delta-mass shifts.
Relative quantification by integrated MS peak area showed major degradants: dimerization (16.8%), succinimide (5.9%), oxidation (4.7%), and desMet (1.4%).
Built-in thresholding (95% purity, 0.5% impurity cutoff) and pass/fail criteria flagged sample purity at 94.2%.

Benefits and practical applications

• End-to-end automation from acquisition to reporting reduces hands-on time and minimizes errors.
• Combined untargeted and targeted workflows accelerate impurity discovery and method refinement.
• Compliance-ready thresholding and systematic quality checks support regulatory documentation.
• High mass accuracy and integrated quantitation facilitate robust peptide quality control in R&D and QA/QC labs.

Future trends and possibilities

Advances in real-time data analytics and AI-driven deconvolution may further accelerate impurity profiling.
Application to larger peptides and protein therapeutics with enhanced high-resolution MS platforms is anticipated.
Integration with automated sampling and cloud-based compliance systems will support high-throughput environments.

Conclusion

The BioAccord LC-MS system and waters_connect Intact Mass Application deliver a flexible, automated workflow for synthetic peptide mass confirmation and impurity profiling.
Automated deconvolution and quantitation simplify compliance-driven analysis and enable rapid characterization of stress-induced degradants.

References

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