Improving Multi-Attribute Method using LC-MS System with Novel Inert Fluidic Pathway

Importance of the topic

Non-specific adsorption of acidic and metal-sensitive peptides to conventional LC fluidic pathways can compromise the sensitivity, accuracy and reproducibility of multi-attribute method (MAM) assays by causing sample loss, peak tailing and variable detector response. Addressing this challenge is essential for reliable quality control in biopharmaceutical development and manufacturing.

Study objectives and overview

This study compares MAM performance on two LC-MS configurations: a conventional stainless steel fluidic path and an advanced pathway featuring MaxPeak High Performance Surface (HPS) technology. The goal is to evaluate improvements in peptide recovery, assay reproducibility and new peak detection sensitivity when using the inert HPS fluidic path.

Used methodology and instrumentation

Samples of NISTmAb were analyzed under controlled and stress conditions using parallel workflows:

• System 1: BioAccord™ UPLC/TOF MS with standard stainless steel fluidic path and ACQUITY UPLC Peptide CSH C18 column (p/n 186006938).
• System 2: BioAccord UPLC/TOF MS paired with ACQUITY Premier UPLC and MaxPeak™ HPS fluidic path, using ACQUITY Premier Peptide CSH C18 column (p/n 186009489).
• Mobile phases: Water/0.1% FA (A) and Acetonitrile/0.1% FA (B); gradient from 1% to 85% B over 100 min at 0.2 mL/min.
• MS settings: ESI+ ionization, m/z 50–2000, capillary voltage 1.2 kV, cone voltage 20 V, collision energy 60–120 V.

Main results and discussion

Comparison of peptide attributes and new peak detection revealed:

• Acidic peptide recovery (GFYPSDIAVEWESNGQPENNYK and deamidated forms) increased significantly on the HPS system, with more symmetrical peaks and higher base peak intensities.
• Relative standard deviation (%RSD) for critical quality attribute (CQA) measurements was reduced or comparable on the HPS pathway, indicating improved assay precision.
• New peak detection sensitivity, assessed by %base peak response for an oxidation peptide (DMIFNFYFDVWGQGTTVTVSSASTK), more than doubled on System 2, demonstrating enhanced detection of low-level modifications.

Benefits and practical application

The integration of MaxPeak HPS fluidic technology into the BioAccord LC-MS platform yields:

• Improved peptide recovery and chromatographic performance for metal-sensitive analytes.
• Enhanced reproducibility and lower variability in quantitative MAM assays.
• Greater sensitivity for new peak detection, supporting robust monitoring of biotherapeutic modifications.

Future trends and opportunities

Advancements in inert fluidic pathway materials and surface chemistries are expected to further reduce adsorption effects and extend the applicability of MAM workflows. Integration with high-resolution MS and automation could enable fully unattended, high-throughput characterization of complex biologics in development and QC environments.

Conclusion

The use of MaxPeak HPS technology in LC-MS multi-attribute methods significantly mitigates non-specific adsorption, enhances peptide recovery, improves assay precision and increases sensitivity for detecting low-level modifications. The BioAccord platform with ACQUITY Premier UPLC and HPS fluidics offers a robust, flexible solution for biopharmaceutical analytics across R&D and quality operations.

Reference

• Ranbaduge N., Birdsall R.E., Yu Y.Q., Chen W. Improving Multi-Attribute Method using LC-MS System with Novel Inert Fluidic Pathway. Waters Corporation, 2021.