Utilizing Empower™ Software to Streamline and Automate the Detection of Sample-to- Sample Differences within Peptide Maps of Biopharmaceuticals

Importance of the Topic

Peptide mapping is a fundamental analytical approach in the development and quality control of biopharmaceutical proteins. It provides detailed information on sequence coverage, post-translational modifications, and degradation products. Automated and streamlined data analysis of peptide maps accelerates decision making, improves reproducibility, and reduces human error in workflows ranging from process development to manufacturing.

Objectives and Study Overview

This study demonstrates how a single chromatography data system can be used to automate the detection of sample-to-sample differences in peptide maps of biotherapeutics. Using infliximab forced-degradation samples, the goals were to:

• Enable rapid, binary comparison of stressed versus reference peptide maps
• Implement custom data fields to flag significant changes or new peaks
• Transfer identified critical quality attributes to a high-throughput QC method

Methodology and Instrumentation

Sample Preparation:

• Infliximab subjected to 0, 1, and 2 weeks of thermal stress at 37 °C
• Reduction, alkylation, desalting, tryptic digestion, and acidification
• Final peptide concentration 0.16 μg/μL

LC-UV/MS Conditions:

• ACQUITY Premier UPLC BSM with tunable UV detector (214 nm) and QDa mass detector
• Premier CSH C18 column (2.1 × 100 mm, 1.7 μm) at 60 °C
• Gradient methods: 80 min for forced degradation; 15.5 min for targeted QC
• Flow rate 0.20 mL/min; injection volume 10 μL

Data Processing:

• Empower 3 CDS used to define custom fields for peak area ratio and flagging rules
• Binary comparison plots provided side-by-side overlays of chromatograms
• Impurity monitoring workflows configured to report against ICH thresholds

Main Results and Discussion

Reproducibility and Sensitivity:

• Retention time RSD average 0.06% without alignment tools
• UV quantitation down to 10 pg mass loads; MS detection extended dynamic range for low-abundance species

Automated Comparison:

• Custom fields automatically calculated fold changes and flagged increases (>2×), decreases (<0.5×), new or missing peaks
• Binary plots visually highlighted seven peptides with significant changes and seven novel degradation products after one week of stress

Targeted QC Method:

• Condensed gradient resolved two peptides and their oxidized forms in 15.5 min
• Retention time RSD 0.16 s; area RSD ~1%
• Impurity tab summarized percent adjusted area and flagged exceedances per ICH guidelines

Benefits and Practical Applications

• Single-platform workflow reduces data exports and manual interventions, improving compliance
• Customizable reports accelerate review and decision making in development and QC
• High-throughput targeted methods enable rapid monitoring of critical quality attributes

Future Trends and Applications

Integration of peptide mapping data with machine learning algorithms may further enhance pattern recognition of degradation products. Expansion of automated workflows to include real-time monitoring and cloud-based collaboration could streamline multi-site method transfers. Adoption of advanced detectors and data-processing tools will continue to drive sensitivity and throughput in both upstream development and downstream QC environments.

Conclusion

This work illustrates how a unified chromatography data system, coupled with a robust UPLC-UV/MS platform, can automate and accelerate peptide map comparisons in biopharmaceutical workflows. Custom fields and impurity monitoring tools enable rapid identification of critical quality attributes and seamless method migration from development to manufacturing.

References

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3. Birdsall RE, McCarthy SM. Increasing Specificity and Sensitivity in Routine Peptide Analyses Using Mass Detection with the ACQUITY QDa Detector. Waters Application Note. 2015;720005377.
4. Koshel BM, Birdsall RE, Yu YQ. LC-UV Based Synthetic Peptide Impurity Tracking and Reporting with Empower 3 Software. Waters Application Note. 2017;720005968.
5. Koshel BM, Birdsall RE, Yu YQ. Using Empower 3 Software for Monitoring Synthetic Peptide Impurities with an ACQUITY QDa Detector. Waters Application Note. 2017;720005967.