Method Transfer and Reliability of the ACQUITY Arc for Peptide Mapping

Significance of the Topic

Peptide mapping is a cornerstone analytical assay in the biopharmaceutical industry for verifying protein identity, characterizing post-translational modifications, and ensuring batch-to-batch consistency. As laboratories adopt modern LC platforms, demonstrating seamless transfer of established methods and consistent performance across instruments is critical to maintain product quality, streamline workflows, and meet regulatory expectations.

Objectives and Study Overview

This study evaluates the equivalency and reproducibility of a 60-minute infliximab peptide mapping method when transferred from an Agilent 1100 Series HPLC System to the Waters ACQUITY Arc System. Key goals include:

• Demonstrate method transfer without altering chromatographic parameters
• Assess system-to-system reproducibility across two ACQUITY Arc instruments
• Incorporate orthogonal mass detection using the ACQUITY QDa Detector

Methodology and Instrumentation

Infliximab samples (10 mg/mL) were reduced, alkylated, and digested with trypsin (1:20 ratio) at 37 °C for 18 hours. Digests (0.4 mg/mL) were injected (75 μL) onto XBridge BEH C18 columns under a linear gradient (5–95% acetonitrile with 0.1% TFA) at 0.5 mL/min and 40 °C. The same method was first run on the Agilent 1100 Series HPLC with DAD detection, then transferred directly to the ACQUITY Arc System using Path 1 and Gradient SmartStart Technology to compensate for dwell volume differences. Two Arc systems, each equipped with a 30 cm preheater, UV/Vis detector (2489) and optional QDa mass detector, were compared. A second set of runs employed smaller (2.5 μm) particles to exploit UHPLC separation capabilities on the same 60-minute gradient.

Main Results and Discussion

• Method Transfer Equivalency: Chromatograms from the Agilent 1100 and ACQUITY Arc systems showed near-identical peak patterns. Relative retention time differences (ΔRRT) for 52 major peaks were under 0.015, confirming effective transfer without parameter changes.
• System-to-System Reproducibility: Two independently prepared Arc systems delivered highly consistent maps, with ΔRRT values below 0.006 across all peaks.
• Enhanced Resolution with UHPLC: Switching to a 2.5 μm column on the Arc System increased peak capacity and improved resolution while maintaining the same run time.
• Mass Confirmation: The ACQUITY QDa Detector provided base peak mass confirmation of individual peptides (e.g., complementary determining region peptide at m/z 897.9), reinforcing identity verification and supporting quality control workflows.

Benefits and Practical Applications

• Facilitates direct transfer of existing HPLC peptide mapping assays to a modern LC platform without revalidation of gradient or temperature conditions.
• Ensures reliable, reproducible results across multiple instruments and sites, supporting global method harmonization.
• Combines routine optical detection with mass confirmation to strengthen confidence in peptide identity during batch release, stability studies, and lot comparisons.
• Maintains flexibility to perform HPLC and UHPLC separations on a single system, optimizing laboratory throughput and resource utilization.

Future Trends and Potential Applications

Advances in LC instrumentation and software will continue to drive integration of high-resolution separations with automated mass detection and data analysis. Anticipated trends include:

• Expanded use of compact mass detectors for in-line peptide and protein characterization.
• Automated method optimization leveraging machine learning to tailor gradients and column selection.
• Cloud-based data management and cross-laboratory comparability tools for global QC harmonization.
• Integration with online digestion platforms for rapid, high-throughput peptide mapping.

Conclusion

The ACQUITY Arc System successfully emulates legacy HPLC peptide mapping methods and enables seamless transition to UHPLC separations, all on a single platform. Method transfer from an Agilent 1100 Series HPLC System was achieved without parameter adjustments, and system-to-system reproducibility was demonstrated across two Arc instruments. Incorporation of the ACQUITY QDa Detector adds orthogonal verification of peptide identity, enhancing confidence in quality control assays.

References

1. Koshel BM, McCarthy SM. Transfer of an SEC Method for Monoclonal Antibody Analysis from HPLC to UHPLC using the ACQUITY Arc System. Waters Application Note 2015. 720005510en.
2. Koshel BM, McCarthy SM. IEX Method Transfer: Replicating a Method for Monoclonal Antibody Analysis on an ACQUITY Arc System. Waters Application Note 2015. 720005529en.
3. Luo Y et al. Peptide Mapping. In: Ahuja S, Scypinski S, eds. Handbook of Modern Pharmaceutical Analysis. Academic Press; 2010:283–359.
4. USP <1055> Biotechnology-Derived Articles – Peptide Mapping. USP38-NF33; 2015.
5. Hong P, McConville PR. Method Transfer from Agilent 1100 Series LC System to the ACQUITY UPLC H-Class System: The Effect of Temperature. Waters Application Note 2015. 720005204en.
6. Waters. ACQUITY Arc System Brochure. Waters Publication 2015. 720005393en.