Improved Analysis of Intact Proteins and Peptides by Reversed Phase HPLC Using the Altura Ultra Inert PLRP-S 1000 Å Column

Improved Analysis of Intact Proteins and Peptides by Reversed-Phase HPLC Using the Altura Ultra Inert PLRP-S 1000 Å Column

Significance of the topic

Reversed-phase liquid chromatography coupled to mass spectrometry (RP-LC/MS) is an essential analytical approach in biopharmaceutical development and quality control for intact proteins, peptides and antibody–drug conjugates (ADCs). Reliable intact-mass analysis informs on molecular weight, post-translational modifications (PTMs), proteoforms and critical quality attributes such as drug-to-antibody ratio (DAR). Minimizing nonspecific adsorption and metal-induced interactions in the LC flow path is crucial to maximize recovery, resolution and mass-spectral quality for these chemically complex analytes.

Study aims and overview

This application note evaluates the analytical benefits of Agilent’s Altura Ultra Inert PLRP-S 1000 Å column hardware compared with conventional stainless-steel PLRP-S 1000 Å columns for intact biomolecule separations. Objectives were to (1) quantify passivation time and analyte recovery across a range of molecules (mAbs, ADCs, small peptides, small globular proteins), (2) assess effects on MS spectral quality and PTM/DAR determination, and (3) demonstrate practical LC/MS conditions compatible with routine intact-mass workflows.

Methodology and experimental design

- Sample set: formulated monoclonal antibodies (mAbs), the ADC sacituzumab govitecan, exenatide (GLP-1), and lysozyme. Samples prepared at 1 mg/mL in 0.1% formic acid; injections typically 1 µL at 0.1 mg/mL unless noted.

- Chromatography: Agilent PLRP-S 1000 Å, 2.1 × 50 mm, 5 µm particle size; comparison between standard stainless-steel hardware and Altura Ultra Inert coated hardware. Mobile phases were 0.1% formic acid (A) and ACN + 0.1% formic acid (B). Typical flow 0.21 mL/min, column temperature 65 °C, 10 min total runtime with a fast gradient program tailored for intact proteins.

- Mass spectrometry: Agilent 6530 Q-TOF operated in positive ion mode with source and gas settings optimized for desolvation and intact-mass detection. Data processed with Agilent MassHunter software and DAR calculator.

Used instrumentation

• Agilent 1290 Multicolumn Thermostat (G7116B)
• Agilent 1290 Bio Multisampler (G7137A)
• Agilent 1290 Bio High-Speed Pump (G7132A)
• Agilent 6530 Q-TOF MS
• Agilent MassHunter Workstation and Qualitative Analysis software; Agilent DAR calculator

Key results and discussion

- Passivation and recovery: Altura Ultra Inert hardware dramatically reduced column passivation time. A representative ADC (sacituzumab govitecan) reached stable signal intensity by the third injection on Altura hardware, whereas stainless-steel hardware required nearly 20 injections to approach the same signal. Overall recovery gains were molecule-dependent:

• Intact mAbs: modest but consistent recovery improvement (~5–10% higher peak area).
• ADCs: ~10% greater recovery versus stainless steel under tested conditions; improved ability to monitor heavy and light chains for DAR calculation.
• Small peptides (e.g., exenatide): large gains in recovery (up to ~2×, i.e., ~100% improvement relative to stainless steel).
• Small globular proteins (e.g., lysozyme): substantial improvement (Altura showed ~15% higher recovery in examples provided).

- Spectral quality and PTM identification: Improved recovery on Altura hardware translated into higher-quality deconvoluted mass spectra, enabling clearer identification of proteoforms and PTMs. Example: atezolizumab spectra resolved species corresponding to loss of lysine residues (–128 and –256 Da). For sacituzumab govitecan, deconvoluted spectra revealed a 418 Da mass loss consistent with loss of the SN-38 payload in some chains; improved signal facilitated reliable DAR quantification (validated DAR ~7.6 reported in literature). Mass accuracy for heavy and light chains was reported around 28 ppm in the example shown.

- Practical observations: The Altura Ultra Inert coating minimizes nonspecific interactions between negatively charged analyte sites and exposed metal ions in stainless-steel housings, thereby reducing initial sample passivation needs and cumulative signal loss. The PS/DVB-based PLRP-S stationary phase (1000 Å) also contributes by reducing silanol-related adsorption compared with silica-based phases and providing large-pore access suitable for high-molecular-weight species.

Benefits and practical applications

• Faster method readiness: substantially reduced passivation time decreases column conditioning cycles and sample consumption prior to routine analysis.
• Higher sensitivity: improved recovery increases signal-to-noise for intact-mass measurements, benefiting low-abundance PTM detection and more accurate deconvolution.
• More reliable DAR and PTM analysis: better recovery and spectral quality improve quantitation and identification of critical attributes for ADCs and mAbs.
• Versatile workhorse: the combination of large-pore PLRP-S chemistry with Ultra Inert hardware suits a broad range of biotherapeutics from small peptides to intact antibodies and ADCs.

Future trends and potential applications

- Wider adoption of inert column housings and polymeric stationary phases is likely as intact mass analysis becomes routine in QC and development, particularly where metal-sensitive analytes (oligonucleotides, phosphorylated proteins, acidic proteins) are analyzed.
- Integration with high-resolution MS and streamlined software tools (automated deconvolution and DAR calculators) will further reduce time-to-result and improve robustness of CQA monitoring.
- Opportunities exist to combine inert hardware with emerging chromatographic chemistries and mobile-phase additives optimized for top-down proteomics and intact-biotherapeutic characterization.

Conclusion

The Altura Ultra Inert PLRP-S 1000 Å column hardware meaningfully improves intact-protein and peptide RP-LC/MS workflows by reducing nonspecific metal-induced adsorption, shortening passivation time, and increasing analyte recovery across a range of biotherapeutics. These gains translate to improved MS spectral quality and more reliable PTM and DAR determinations, making the Altura PLRP-S 1000 Å a practical option for routine intact-mass characterization in both development and QC laboratories.

References

1. Donnelly, D. P.; et al. Best Practices and Benchmarks for Intact Protein Analysis for Top-Down Mass Spectrometry. Nature Methods 2019, 16(7), 587–594. doi:10.1038/s41592-019-0457-0.
2. McCalley, D. V.; Guillarme, D. Evaluation of Additives on Reversed-Phase Chromatography of Monoclonal Antibodies Using a 1000 Å Stationary Phase. Journal of Chromatography A 2020, 1610, 460562. doi:10.1016/j.chroma.2019.460562.
3. Kopp, A.; et al. Antibody–Drug Conjugate Sacituzumab Govitecan Drives Efficient Tissue Penetration and Rapid Intracellular Drug Release. Molecular Cancer Therapeutics 2022, 22(1), 102–111. doi:10.1158/1535-7163.mct-22-0375.