AdvanceBio HIC: a Hydrophobic HPLC Column for Monoclonal Antibody (mAb) Variant Analysis

Importance of the topic

Oxidation of monoclonal antibodies is a critical degradation pathway that can reduce therapeutic efficacy and shelf life. Monitoring and characterizing oxidized variants ensures product quality and safety in biopharmaceutical development and QA/QC processes.

Objectives and overview of the study

This application note demonstrates the use of the Agilent AdvanceBio HIC column on the Agilent 1260 Infinity II bio-inert LC system to separate oxidized monoclonal antibody variants from their native form. Two chemical oxidants (t-butyl hydroperoxide and hydrogen peroxide) are compared, and optimal chromatographic conditions are established.

Methodology and instrumentation

• Instrumentation
  Agilent 1260 Infinity II bio-inert pump, multisampler with cooler, column thermostat with bio-inert heat exchanger, and diode array detector with bio-inert flow cell (OpenLab CDS 2.2).
• Column
  AdvanceBio HIC, silica-based, 4.6 × 100 mm.
• Mobile phases
  A: 50 mM sodium phosphate, pH 7.0; B: 2 M ammonium sulfate in 50 mM sodium phosphate, pH 7.0.
• Oxidation protocols
  • t-BHP treatment: 0.2 % (v/v) 70 % t-butyl hydroperoxide, 24 h at room temperature.
  • H2O2 treatment: 0.2 % (v/v) 50 % hydrogen peroxide, 24 h at room temperature.
  Samples were buffer-exchanged and concentrated to 1 mg/mL using 10 kDa centrifugal filters before analysis.
• Chromatographic conditions
  Standard gradient: start 50 % A/50 % B, linear to 100 % A over 20 min at 0.5 mL/min, then re-equilibrate.
  Shallow gradient: start 40 % A/60 % B, linear to 90 % A over 40 min at 0.3 mL/min for enhanced resolution.

Main results and discussion

Under standard conditions, t-BHP oxidation produced multiple early-eluting peaks (labeled 1–6) corresponding to surface-accessible methionine sulfoxide species, while the native antibody eluted as a single peak at longer retention. H2O2 induced more extensive oxidation, yielding three predominant early peaks and indicating deeper methionine modification. A shallower gradient at lower flow rate improved separation of individual oxidized variants within a reasonable analysis time.

Time-course monitoring of t-BHP oxidation showed progressive peak multiplication and broadening, with extended incubation (10 h) leading to a broad, shifted peak likely due to partial unfolding from buried methionine oxidation.

Benefits and practical applications

The AdvanceBio HIC column allows direct analysis of intact mAbs under physiological pH without subunit digestion or harsh conditions. This approach preserves native conformation and simplifies sample preparation. It is suitable for stability studies, forced-degradation screening, and quality control of biotherapeutics including antibody-drug conjugates and bispecific formats.

Future trends and potential applications

Emerging directions include coupling HIC separations with high-resolution mass spectrometry for site-specific variant identification, development of tailored bonded phases for enhanced selectivity, automation of high-throughput variant screening, and application to complex constructs such as ADCs and multispecific antibodies.

Conclusion

The Agilent AdvanceBio HIC column on the 1260 Infinity II bio-inert LC system provides high-resolution separation of oxidized mAb variants from native forms. Optimized conditions using shallow gradients and controlled flow rates enable detailed characterization of oxidation-induced hydrophobicity changes without extensive sample preparation.

References

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