The BioAccord System With ACQUITY Premier for Improved Peptide CQA Monitoring

Importance of the Topic

Peptide-based multi-attribute methods (MAM) using LC-MS are transforming biotherapeutic quality assessment by enabling direct measurement of multiple critical quality attributes (CQAs) in a single assay. Robust chromatographic performance and consistent mass spectrometric response are essential to ensure accurate quantitation, particularly for low-abundance or metal-sensitive peptides that are prone to adsorption and signal loss on conventional stainless-steel surfaces.

Study Objectives and Overview

This application note compares the performance of a standard BioAccord System equipped with stainless-steel flow paths to the BioAccord System configured with the ACQUITY Premier UPLC featuring MaxPeak High Performance Surfaces (HPS) Technology. The primary goals were to evaluate improvements in acidic peptide recovery, assay sensitivity, reproducibility across a 3-order magnitude dynamic range, and overall suitability for peptide MAM workflows.

Methodology and Instrumentation

Sample preparation and chromatographic conditions were held constant while comparing two configurations:

• BioAccord with ACQUITY UPLC I-Class PLUS (stainless-steel flow path)
• BioAccord with ACQUITY Premier UPLC (inert MaxPeak HPS surfaces)

Both systems used CSH C18 columns matched to each flow path, operated at 60 °C with 0.1% formic acid in water (A) and acetonitrile (B). Detection employed a tunable UV detector and ACQUITY RDa mass spectrometer in positive ESI mode (m/z 50–2000). Data processing and attribute monitoring were performed using waters_connect with the Peptide MAM App and LC-MS Tool Kit.

Main Results and Discussion

The ACQUITY Premier configuration delivered at least a two-fold increase in MS signal intensity for acidic peptides such as the PENNY fragment from NISTmAb, revealing previously undetectable deamidation species. Improved recovery translated into richer b/y fragmentation spectra and enhanced confidence in automated peak assignments. When monitoring a panel of CQAs over 0.1–2.0 µg loadings, the Premier system maintained area-% reproducibility (%RSD ≤ 20% for most attributes), with sub-10% RSD for oxidation modifications. A linear dynamic range from 0.1 to 1.0 µg (R2=0.99) was established, enabling detection of glycopeptides at 0.08% relative abundance with %RSD ≈ 3% across five injections.

Benefits and Practical Applications

• Enhanced recovery and peak shape for acidic, metal-sensitive peptides
• Stable MS response at both high and low loading levels
• Lower %RSD for monitored CQAs, improving assay precision
• Broad spectral dynamic range spanning three orders of magnitude
• Seamless integration with compliant-ready waters_connect informatics for automated workflows

Future Trends and Opportunities

Advances in inert surface chemistries and UHPLC hardware will continue to reduce analyte adsorption, enabling further sensitivity gains. Integration with real-time data analytics and expanded informatics capabilities may facilitate adaptive method optimization and deeper characterization of low-level modifications. The principles demonstrated here can be extended to other PTMs, host-cell proteins, and complex sample matrices in biopharmaceutical development.

Conclusion

The BioAccord System paired with ACQUITY Premier and MaxPeak HPS Technology offers a robust, high-performance platform for peptide MAM assays. By minimizing metal-surface interactions, it achieves superior peptide recovery, reproducibility, and dynamic range without altering existing RPLC-MS conditions, making it ideally suited for routine QC, method transfer, and lifecycle management of protein therapeutics.

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