LC-MS analysis of intact and subunit-level mAb enabled by a novel monodisperse supermacroporous reversed-phase platform

Significance of the topic

Monoclonal antibodies are structurally complex biotherapeutics whose quality and efficacy depend on precise characterization at multiple structural levels. Fast, MS-compatible chromatographic methods that can handle intact proteins and enzymatically generated subunits reduce assay complexity, accelerate decision making during development and quality control, and minimize the need for multiple specialized columns. The study evaluated a novel monodisperse supermacroporous reversed-phase stationary phase for unified LC–MS analysis of intact mAbs and common subunits, addressing a practical bottleneck in biopharmaceutical analytics.

Objectives and overview of the study

• Evaluate a single reversed-phase column (SurePac Protein RP MDi) based on a 2.5 µm monodisperse supermacroporous (SMP) polymeric phase for unified analysis of monoclonal antibodies at intact, reduced and IdeS-digested (subunit) levels.
• Demonstrate rapid, LC–MS compatible separations (10-min gradient) that provide baseline separation of intact mAb, light chain (LC), heavy chain (HC), single-chain Fc (scFc) and F(ab′)2 with accurate mass determination.
• Assess mass accuracy and identify dominant proteoforms of the NISTmAb test material as a representative case study.

Methodology

• Stationary phase: 2.5 µm monodisperse supermacroporous polymeric stationary phase (SurePac Protein RP MDi), column 2.1 × 50 mm.
• Chromatography: Reversed-phase gradients using 0.1% formic acid in water (Eluent A) and MeCN/H2O/FA (90:9.9:0.1, Eluent B). Typical flow 0.4 mL/min, column/chamber temperature 80 °C, active pre-heater enabled, injection volumes 0.25–0.5 µL depending on sample and format. A 10–18 min program was used with the critical separations obtained in a 10-min gradient window.
• Sample preparation: Intact NISTmAb analyzed directly. Subunits generated by DTT reduction (20 mM DTT, 30 min, 37 °C, 300 rpm) for LC/HC and by IdeS digestion (1 unit enzyme per µg mAb, 30 min, 37 °C, 300 rpm) for scFc and F(ab′)2.
• Mass spectrometry: Thermo Scientific Orbitrap Exploris 480 with OptaMax NG ion source in positive ion protein mode using HESI. Full MS scans (single and two‑segment methods) adjusted for m/z ranges appropriate for intact and subunit analyses. Data processing used Xcalibur, Thermo Freestyle and BioPharma Finder with Xtract and ReSpect deconvolution algorithms.

Used instrumentation

• UHPLC: Thermo Scientific Vanquish Horizon UHPLC system.
• Mass spectrometer: Thermo Scientific Orbitrap Exploris 480 with OptaMax NG ion source.
• Column: Thermo Scientific SurePac Protein RP MDi, 2.1 × 50 mm, 2.5 µm SMP polymeric stationary phase.
• Software: Thermo Xcalibur 4.7, Freestyle 1.8, BioPharma Finder 5.4 (ReSpect and Xtract algorithms).

Main results and discussion

• The SurePac Protein RP MDi column achieved baseline separation of intact mAb and common subunits (LC, HC, scFc, F(ab′)2) within a 10-min gradient under LC–MS compatible conditions, enabling a single, fast workflow for multilevel mAb characterization.
• High-resolution and lower-resolution deconvolution (Xtract, ReSpect) produced accurate mass assignments for dominant proteoforms. Measured average masses for intact and subunit species agreed with theoretical values within a few ppm (typical Δ in the range of ~−2.5 to +4.4 ppm reported for selected species), consistent with confident proteoform assignment by mass agreement.
• Observed dominant modifications in the NISTmAb included C‑terminal lysine truncation and N‑terminal pyroglutamate; scFc glycoforms A2G0F, A2G1F and A2G2F were identified as the principal glycoforms based on mass differences. These assignments are putative and recommended for orthogonal confirmation (e.g., LC–MS/MS peptide mapping) when site-specific identification is required.
• Chromatograms at the intact level showed a major peak with limited resolution of low-abundance proteoforms, which is expected because intact-level separations compress heterogeneity; however, the chromatographic performance provided high-quality spectra for deconvolution and mass determination.
• Operating at elevated temperature (80 °C) and using the SMP phase promoted fast mass-transfer and access to both large and smaller protein analytes, enabling a compromise between resolving power and speed across size ranges.

Benefits and practical applications

• Single-column workflow: Reduces need for multiple specialized columns for intact and subunit analyses, simplifying method setup, lowering instrument downtime and saving bench time.
• Speed and throughput: Ten-minute gradients allow rapid screening of mAb variants and routine QC-style checks where mass confirmation and relative variant quantitation are required.
• MS compatibility: Use of volatile formic acid and organic solvent mixtures enables direct coupling to high-resolution MS for accurate mass measurement and deconvolution-based proteoform assignment.
• Subunit-level capability: Effective separation of IdeS and reduction products aids localization of major glycoforms and common PTMs without full peptide mapping, offering a faster intermediate-level characterization approach.
• Analytical robustness: Uniform particle size and supermacroporous morphology improve mass transport and reproducibility for a wide analyte size range (small subunits to intact mAbs).

Limitations and practical considerations

• Intact-level resolution is limited for low-abundance proteoforms; peptide mapping or targeted MS/MS is still necessary for site-specific PTM localization and quantitation of very low-level variants.
• High column temperature (80 °C) may risk heat-induced modifications or conformational changes for some labile analytes; method validation should evaluate potential artifacts.
• Glycoform assignments based solely on mass are putative—orthogonal fragmentation or enzymatic/chemical confirmation improves assignment confidence.
• Sample load, injection volume and concentrations need optimization for specific instruments and detector sensitivities to avoid ion suppression or detector saturation.

Future trends and potential uses

• Broader adoption of unified stationary phases for multi-level mAb analysis in development and QC, reducing method multiplicity and increasing throughput.
• Integration with automated sample preparation and online digestion workflows to create high-throughput pipelines for variant screening.
• Combination with targeted MS/MS or native MS approaches to enhance structural characterization while maintaining rapid chromatographic screening.
• Expansion of SMP-type polymeric phases to other classes of biologics (fusion proteins, ADCs, multispecifics) and tailoring pore architectures for even larger assemblies.
• Regulatory uptake for lot release and stability screening where rapid, reproducible mass confirmation is adequate; concomitant development of qualification guidelines for such unified assays.

Conclusion

The study demonstrates that a monodisperse supermacroporous reversed-phase column (SurePac Protein RP MDi) can provide a fast, MS-compatible single-column workflow for intact and subunit-level mAb characterization. Baseline separation of LC, HC, scFc and F(ab′)2 within a 10-min gradient and accurate mass determination of dominant proteoforms were achieved on a Vanquish–Orbitrap platform. While intact-level heterogeneity resolution remains inherently limited, the approach delivers a practical, high-throughput solution for routine proteoform screening and subunit-level glycoform assessment, with orthogonal techniques recommended for definitive site-specific confirmation.

Reference

• Žvirblis A., Dembovskytė O., Ma K., Mitterer C., De Pra M., Steiner F. LC–MS analysis of intact and subunit-level mAb enabled by a novel monodisperse supermacroporous reversed-phase platform. Thermo Fisher Scientific application note. 2026. (Instrumentation: Vanquish Horizon UHPLC; Orbitrap Exploris 480; SurePac Protein RP MDi column.)