Using Hydrophilic Interaction Chromatography for Heightened Product Characterization to Overcome Challenges with Hydrophobic Monoclonal Antibodies and Antibody Drug Conjugates

Importance of the Topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) are cornerstone biotherapeutics whose structural integrity and subunit profiles critically influence efficacy, safety, and regulatory approval. Conventional reversed-phase liquid chromatography (RPLC) often struggles with incomplete recovery of hydrophobic domains and on-column degradation, complicating accurate characterization.

Study Objectives and Overview

This study evaluates hydrophilic interaction chromatography (HILIC) as an orthogonal separation technique for IdeS-derived mAb subunits (light chain, Fd′, scFc) and ADC conjugates. Key aims include comparing chromatographic recovery, resolution of glycoforms and payload isomers, temperature dependence, and artifact formation between HILIC and RPLC.

Methods and Instrumentation Used

• Sample Preparation: Therapeutic mAb and prototype ADC samples were digested with IdeS enzyme at pH 6.6, 37 °C, followed by denaturation and reduction with guanidine hydrochloride and DTT.
• HILIC Conditions: ACQUITY UPLC Glycoprotein BEH Amide 300 Å, 1.7 µm, 2.1 × 150 mm column on a Waters ACQUITY UPLC H-Class Bio System; mobile phases of acetonitrile–water with 0.05–0.1 % TFA; column temperature range 30–80 °C; flow rate 0.2 mL/min.
• RPLC Conditions: ACQUITY UPLC Protein BEH C4 300 Å, 1.7 µm, 2.1 × 100 mm column; water–acetonitrile gradients with 0.1 % TFA; column temperatures up to 85 °C.
• Detection: UV absorbance at 214 nm with optional online high-resolution mass spectrometry for accurate mass confirmation.

Key Results and Discussion

• Orthogonal Selectivity: HILIC retention via partitioning into a hydrated layer complements RPLC’s hydrophobic adsorption, reversing elution order of subunits and glycoforms.
• Enhanced Recovery and Lower Temperature Dependence: HILIC achieved quantitative recovery of all mAb subunits at temperatures as low as 50 °C without detectable hydrolysis artifacts, whereas RPLC required 85 °C and still exhibited on-column degradation peaks.
• ADC Subunit Analysis: HILIC maintained consistent recovery and resolved positional isomers in payload-loaded Fd′ species, while RPLC retention decreased upon conjugation, risking loss of hydrophobic species.

Benefits and Practical Applications

• Minimized on-column degradation and artifact formation in protein subunit profiling.
• Reliable quantitative recovery of hydrophobic mAb domains and ADC payloads under milder conditions.
• Simplified integration with LC–MS workflows for detailed glycoform and conjugate mapping.
• Adaptable to quality control and method development in biopharmaceutical laboratories.

Future Trends and Possibilities for Use

• Application of HILIC to other complex biotherapeutics such as fusion proteins, bispecifics, and pegylated antibodies.
• Coupling HILIC with multi-dimensional separation strategies for comprehensive structural characterization.
• Development of novel HILIC phases tailored for enhanced selectivity of specific post-translational modifications.

Conclusion

HILIC offers a robust, complementary approach to RPLC for mAb and ADC subunit analysis, delivering superior recovery, reduced artifact formation, and effective resolution of glycoforms and payload variants. Its compatibility with lower temperatures and mass spectrometry makes it an invaluable tool for biopharmaceutical characterization and quality control.

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