Improving the Recovery of Intact Antibodies from Reversed-Phase Separations Using a BioResolve RP mAb Polyphenyl Column

Importance of the Topic

Monoclonal antibodies are critical biotherapeutics with complex structures that require precise analytical characterization to ensure safety and efficacy.
Reversed-phase liquid chromatography (RPLC) offers high separation efficiency but intact mAb analysis often suffers from limited recovery and sample degradation at high temperatures and with strong ion-pairing reagents.

Objectives and Study Overview

This study aimed to demonstrate the improved recovery of intact antibodies across a range of temperatures using a silica-based, solid-core BioResolve RP mAb Polyphenyl Column.
A direct comparison was made against a conventional polymeric column to assess recovery, resolution, and applicability for multiple therapeutic mAbs.

Methodology and Instrumentation

• Instrument: ACQUITY UPLC H-Class Bio System with ACQUITY TUV Detector (λ=280 nm) and Empower 3 Software
• Columns:
  
  • BioResolve RP mAb Polyphenyl, 2.1×50 mm, 2.7 µm, 450 Å solid-core silica
  • Polymeric PS-DVB, 2.1×50 mm, 1500 Å porous particles
• Mobile phases:
  
  • Water and acetonitrile with either 0.1% TFA
  • Water and acetonitrile with 0.1% DFA and 5% n-butanol
• Temperature range: 40–80 °C
• Sample panel: Waters Intact mAb Mass Check Standard, Rituximab, Tocilizumab, Bevacizumab (2.5 µg load)

Main Results and Discussion

Operation at high temperature (80 °C) with 0.1% TFA yielded good resolution but dramatically reduced recovery below 70 °C due to strong adsorption.
Replacing TFA with 0.1% DFA and adding 5% n-butanol restored elution and significantly increased recovery at lower temperatures, preserving resolution of impurity peaks.
Across four mAb samples, the BioResolve column with DFA/butanol showed higher or comparable recovery compared to the polymeric column, demonstrating robustness and sample-to-sample consistency.

Benefits and Practical Applications

• Enables high-recovery intact mAb separations at reduced temperatures, minimizing on-column degradation.
• Extends column lifetime by avoiding harsh conditions.
• Improves method robustness for QC and biopharmaceutical development workflows.

Future Trends and Potential Applications

• Extension of optimized RPLC methods to other large biomolecules such as fusion proteins and antibody-drug conjugates.
• Integration with mass spectrometry for enhanced structural characterization.
• Development of new stationary phases and mobile-phase chemistries to further reduce adsorption.

Conclusion

The BioResolve RP mAb Polyphenyl Column combined with milder ion-pairing additives achieves improved intact antibody recovery at lower temperatures, offering a reliable platform for detailed mAb analysis while preserving sample integrity and column performance.

References

1. Liu JKH. The history of monoclonal antibody development–progress, remaining challenges and future innovations. Ann Med Surg. 2014;3:113–116.
2. Dong M, Hamuro L, Mhatre R, et al. HPLC for characterization and quality control of therapeutic monoclonal antibodies. LCGC North Am. 2014;32(10):796–808.
3. Fekete D, Guillarme D, Carr PW. Impact of mobile phase temperature on recovery and stability of monoclonal antibodies using recent reversed-phase stationary phases. J Sep Sci. 2012;35:3113–3123.
4. Nguyen J, Betancourt P, et al. Designing a new particle technology for reversed phase protein separations through optimization of diffusion properties. Waters Application Note 720006168EN. 2018.
5. Nguyen J, Betancourt P, et al. A novel phenyl bonded phase for improved protein reversed phase separation. Waters Application Note 720006169EN. 2018.
6. Bondarenko PV, Elliott S, et al. Optimization of a reversed-phase LC/MS method for characterizing recombinant antibody heterogeneity and stability. J Chromatogr A. 2006;1120:112–120.