Intact & Subunit Analysis Using Reversed Phase - Agilent BioHPLC Columns Application Compendium

Significance of the topic

Reversed-phase chromatography under denaturing conditions is a key tool for separating proteins based on hydrophobicity and denaturing them for detailed mass measurement. This method concentrates samples on the column, ensures high sensitivity, and when coupled to mass spectrometry provides accurate mass profiles for intact monoclonal antibodies (mAbs) and their subunits. With the increasing importance of therapeutic mAbs and biosimilars, rapid and precise characterization of molecular weight, glycoforms, and charge variants is critical for quality control, comparability studies, and biosimilarity assessment.

Objectives and overview of the study/article

The primary objective is to present integrated workflows for intact and subunit analysis of mAbs, exemplified by the Agilent NISTmAb standard, using reversed-phase chromatography on wide-pore columns. The work highlights:

• Rapid intact mass measurement of mAbs
• On-column or on-cartridge deglycosylation and proteolysis to generate subunit fragments
• High-resolution separation of heavy, light, Fab, Fc fragments
• Automated sample preparation for enhanced throughput and reproducibility

Methodology and instrumentation

Reversed-phase separations were conducted under denaturing conditions using Agilent wide-pore columns (300–450 Å) bonded with C4, SB-C8, diphenyl, or polymeric PLRP-S phases. Agilent 1290 Infinity II UHPLC systems provided gradient control at elevated temperatures (up to 80 °C) with mobile phases containing formic acid or TFA in water, acetonitrile, and isopropanol. Mass analysis employed the Agilent 6545XT AdvanceBio LC/Q-TOF with Dual JetStream source, operating in extended mass range mode (up to 10,000 m/z) and optimized gas and voltage settings. Sample preparation was automated on the Agilent AssayMAP Bravo platform with streptavidin cartridges for antigen or Protein L capture, followed by on-cartridge PNGase F deglycosylation, IdeS proteolysis, and TCEP reduction.

Instrumentation used

• Agilent 1290 Infinity II UHPLC system
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual JetStream source
• Agilent PLRP-S and AdvanceBio RP-mAb columns (C4, SB-C8, diphenyl)
• Agilent AssayMAP Bravo automated sample preparation platform
• Agilent MassHunter BioConfirm software for deconvolution and data analysis

Main results and discussion

High-resolution intact mAb analysis using a 4-minute RP gradient yielded six major glycoform peaks with mass accuracy below 1 ppm for NISTmAb. Automated deconvolution resolved minor heterogeneities such as glycan truncations. An intact ADC analysis detected D0–D8 drug-to-antibody species and calculated an average DAR of 3.5. Subunit workflows combining on-cartridge digestion and reduction achieved clear separation of heavy and light chains, Fab, Fc, and Fd′ fragments within 8 minutes, with mass deviations typically <5 ppm. Mirror-plot comparison of innovator and biosimilar mAb deconvolutions revealed high similarity in glycoform distribution and intact mass.

Benefits and practical applications

• Rapid throughput: entire 96-well plate sample preparation and analysis within a workday
• Reduced human error and improved reproducibility via automation
• High mass accuracy and resolution for intact proteins and subunits
• Flexibility to perform deglycosylation, proteolysis, and reduction in one workflow
• Essential for QC release testing, biosimilar comparability, and ADC characterization

Future trends and applications

Emerging trends include further miniaturization of sample preparation, column technologies with novel ligands for enhanced selectivity, AI-driven method development, and integration with orthogonal techniques such as capillary electrophoresis. Increased adoption of high-throughput LC-MS platforms and automated data analysis will support accelerated biotherapeutic development and regulatory compliance.

Conclusion

The integration of advanced reversed-phase chromatography, high-resolution Q-TOF mass spectrometry, and automated sample preparation delivers a comprehensive and efficient workflow for intact and subunit mass analysis of monoclonal antibodies. This approach ensures rapid, accurate, and reproducible characterization, meeting the evolving demands of biopharmaceutical quality control and comparability studies.

Reference

• Intact Protein Analysis Using an Agilent 6550 Q-TOF Mass Spectrometer, Agilent Technologies, publication 5991-2116EN.
• Analysis of Monoclonal Antibody Using Agilent 1290 Infinity LC and 6530 Accurate-Mass Q-TOF, Agilent Technologies, publication 5991-4266EN.
• An Integrated Workflow for Automated Calculation of Antibody-Drug Conjugate DAR, Agilent Technologies, publication 5991-7366EN.