PEEK-lined columns and bio-inert supplies to complement the Agilent 1290 Infinity II bio LC system

Importance of Topic

Biotherapeutic proteins must be carefully characterized to ensure correct manufacturing, detect trace impurities, and confirm potency. Post-translational modifications (PTMs) and subtle variants can impact safety and efficacy, making robust critical quality attribute (CQA) analysis essential in pharmaceutical development and quality control.

Objectives and Overview of the Study

This application note evaluates the performance of a fully biocompatible liquid chromatography flow path—including PEEK-lined columns and bio-inert consumables—coupled with the Agilent 1290 Infinity II Bio LC system. Key goals are to:

• Characterize PTMs and charge, size, and glycan variants in therapeutic proteins.
• Demonstrate reduced metal-induced artifacts for proteins and phosphorylated analytes.
• Illustrate improved recovery, peak shape, and reproducibility across SEC/MS, reversed-phase, and HILIC methods.

Methodology and Instrumentation

This study employed multiple LC modes and detection techniques to cover intact protein and peptide analyses:

• Agilent 1290 Infinity II Bio LC system with biocompatible flow path and InfinityLab Quick Change valves for automated cleaning and column switching.
• PEEK-lined AdvanceBio columns: SEC (1.9 μm, 200 Å), PLRP-S 1000 Å for intact protein reverse-phase, EC-C18 for peptide mapping, and Poroshell HILIC-Z for nucleotide analysis.
• Detectors: variable-wavelength UV, diode array, fluorescence, and MS detection on an Agilent 6545XT AdvanceBio LC/Q-TOF.
• Sample modes: denaturing (0.1% formic acid/acetonitrile) and native (ammonium acetate) for myoglobin; HILIC-DAD for nucleotides.

Main Results and Discussion

PEEK-lined columns eliminated metal interactions, reducing sample priming and saving time.
SEC/MS of myoglobin provided clear deconvoluted masses for apo- and holo-forms under denaturing and native conditions.
PLRP-S reverse-phase analysis of ramucirumab achieved sharper peaks, improved symmetry (1.84 vs. 2.51), and reduced tailing compared to stainless steel columns.
HILIC-DAD analysis of nucleotides (A, AMP, ADP, ATP) on PEEK-lined HILIC-Z columns yielded higher recovery and consistent retention versus stainless steel hardware.
Ultralow-dispersion capillaries further narrowed peaks, especially at low flow rates in SEC methods.

Benefits and Practical Applications

• Enhanced sensitivity and reproducibility for labile proteins and phosphorylated analytes.
• Reduced method development time by minimizing column priming and metal artifact formation.
• Seamless integration of multiple LC modes on a single platform for routine CQA monitoring in QA/QC and R&D.

Future Trends and Applications

Emerging directions include broader adoption of fully bio-inert flow paths in high-throughput workflows, integration with advanced MS and bioinformatics for deeper PTM profiling, miniaturized systems for single-cell proteomics, and AI-driven method optimization to further accelerate biopharma analytics.

Conclusion

A biocompatible LC flow path built around PEEK-lined columns and bio-inert consumables significantly improves the analysis of therapeutic proteins and phosphorylated compounds. The Agilent 1290 Infinity II Bio LC system, paired with specialized AdvanceBio columns, delivers high sensitivity, robust CQA monitoring, and streamlined workflows for the biopharmaceutical industry.

References

• Sandra K., Vandenbussche J., Vanhoenacker G., t’Kindt R., Sandra P., Krieger S., Schneider S., Huber U. HPLC-DAD Analysis of Nucleotides Using a Fully Inert Flow Path. Agilent Technologies Application Note, 2021.