A Novel Phenyl Bonded Phase for Improved Reversed-Phase Separations of Proteins

Importance of the Topic

Reversed-phase liquid chromatography (RPLC) is a cornerstone technique for characterizing the microheterogeneity of therapeutic proteins such as monoclonal antibodies (mAbs). The complex structure and microvariants of mAbs require high resolution and selectivity to ensure product efficacy and safety. Optimizing stationary phase chemistry, while maintaining compatibility with mass spectrometry, remains a significant challenge in the field.

Objectives and Overview

This work introduces a novel high-coverage phenyl bonded phase on a solid-core, wide-pore silica particle (450 Å, 2.7 µm) designed to improve RPLC separations of intact mAbs and subunits. Key performance metrics—effective peak capacity, selectivity, carryover, acid stability, and temperature/ion-pairing dependence—are compared against established C4, phenyl, and polymeric divinylbenzene (DVB) phases.

Methodology

Samples included a reduced, IdeS-digested NIST mAb subunit standard and infliximab subunits, as well as an intact mAb mass check standard. Separations were performed on 2.1 × 50–150 mm columns at elevated temperatures (up to 90 °C) using gradients from 15–55% organic modifier with either 0.1% trifluoroacetic acid (TFA) or 0.1%/0.05% formic acid (FA) in water and acetonitrile. Effective peak capacities were calculated from retention time spans and average peak widths. Carryover and acid stability were assessed with repeated gradients and prolonged exposure to 0.5% TFA at 60 °C, respectively.

Instrumentation Used

• ACQUITY UPLC H-Class Bio System
• MassLynx 4.1 Software
• UNIFI 1.8 Software

Results and Discussion

Performance testing revealed that the polyphenyl phase consistently delivered 13–50% higher effective peak capacity under TFA conditions and superior resolution of lysine variants compared with benchmark columns. Under FA conditions, the new phase maintained leading performance despite weaker ion pairing. Carryover experiments showed undetectable memory effects (<0.1%), outperforming alternatives with up to 2.5% carryover. Acid stability studies demonstrated only a 20% loss in retention after 21 hours in 0.5% TFA at 60 °C, whereas C4 phases lost binding capacity entirely. Temperature and ion pairing dependence studies indicated that high resolution could be achieved at reduced temperatures (50–70 °C) and lower TFA concentrations (0.02–0.05%), mitigating on-column protein degradation and improving MS compatibility.

Practical Benefits and Applications

The improved selectivity and resolution enable detailed mapping of mAb variants and subunits in biopharmaceutical research and quality control. The phase’s acid stability and low carryover extend column lifetime and analytical reproducibility. Compatibility with lower ion pairing strengths facilitates coupling to mass spectrometry for in-depth proteoform analysis.

Future Trends and Potential Applications

Advances in bonded phase design will likely focus on further masking silanol interactions and extending phase lifetimes under milder conditions. The demonstrated capability to operate at reduced temperature and TFA levels suggests broader adoption in native MS workflows and continuous bioprocess monitoring. Emerging stationary phase chemistries may enable rapid screening of diverse protein therapeutics and expanded use in proteomics.

Conclusion

The BioResolve RP mAb Polyphenyl column, featuring a high-coverage phenyl bonded phase on a solid-core wide-pore silica particle, offers exceptional resolution, selectivity, stability, and low carryover for RPLC of intact antibodies and subunits. Its robustness under varied temperature and ion pairing conditions supports reliable, MS-compatible analyses of complex protein therapeutics.

References

1. Bobály B.; Sipkó E.; Fekete S. J. Chromatogr. B. 2016, 1032, 3–22.
2. Ren D. et al. J. Chromatogr. A. 2007, 1175, 63–68.
3. Nguyen J. M. et al. Waters Application Note 720006168EN, January 2018.
4. Ren D.; Pipes G. D.; Bondarenko P. V.; Treuheit M. J.; Gadgil H. S. J. Chromatogr. A. 2008, 1179, 198–204.
5. Trammell B. C. et al. J. Chromatogr. A. 2004, 1060, 153–163.
6. Faid V.; Leblanc Y.; Bihoreau N.; Chevreux G. J. Pharm. Biomed. Anal. 2018, 149, 541–546.
7. Schiel J. E.; Davis D. L.; Borisov O. V. Biopharmaceutical Characterization: The NISTmAb Case Study; ACS, 2015.
8. Ranbaduge N.; Shion H.; Lauber M. A.; Yu Y. Waters Technology Brief 720006199EN, January 2018.