Intact Protein Analysis in Plasma Using Automated 2D-LC/TOF with Multiple Heart-Cutting

Significance of the topic

Direct intact (native) and top-down analysis of proteins in complex matrices enables detection and characterization of whole proteoforms, post-translational modifications and proteolytic fragments that are often missed by bottom-up workflows. Developing methods that allow intact protein mass analysis directly from plasma without affinity enrichment addresses major analytical challenges in biopharma and translational research: wide dynamic range, severe ion suppression, and the need to preserve native structure for accurate proteoform and drug-conjugate characterization.

Objectives and overview of the study

To demonstrate a non-affinity, orthogonal 2D-LC/TOF workflow with automated multiple heart-cutting for native intact protein analysis directly in human plasma. The study used trastuzumab (a monoclonal antibody) and the antibody–drug conjugate T-DM1 as model analytes to evaluate sensitivity, mass accuracy, preservation of glycoforms, and the ability to resolve ADC drug-to-antibody ratio (DAR) heterogeneity without immunocapture.

Methodology

Key experimental design and sample handling:

• Model analytes: formulated trastuzumab and T-DM1 spiked into neat human plasma at 1 µg/µL and serially diluted to assess sensitivity (down to 62.5 ng/µL).
• Sample cleanup: cold centrifugation (4 °C, 20,000 × g, 20 min) followed by buffer exchange/cleanup through 30 kDa MWCO centrifugal filters into 200 mM ammonium acetate to preserve native conditions and approximate initial plasma volume.
• Separation strategy: two-dimensional liquid chromatography using size-exclusion chromatography (1D SEC) to fractionate intact proteins followed by online ion-exchange chromatography (2D IEX) for orthogonal resolving power. UV-guided and automated multiple heart-cutting isolated SEC fractions into 40 µL loop volumes for targeted high-resolution 2D analysis.
• Mass spectrometry: high-sensitivity, accurate-mass TOF acquisition under native electrospray conditions; data processed using MassHunter acquisition and BioConfirm software to extract native charge-state spectra and deconvoluted masses.

Used instrumentation

Instrumentation explicitly employed in the workflow:

• Agilent 1290 Infinity III 2D-LC platform with Bio Multisampler, Multicolumn Thermostat, Diode Array Detector (Max-Light cell), Bio Flexible and Bio High-speed Pumps, valve drives configured for automated multi-heart-cutting and loop storage (40 µL bio loops), and a diverter valve.
• Columns: AdvanceBio SEC (300 Å, 2.7 µm, 4.6 × 300 mm) as 1D; Agilent Bio WAX NP5 (2.1 × 250 mm) as 2D IEX.
• Mobile phases: ammonium acetate buffers (200 mM for SEC; 10–500 mM AA for IEX gradient) to maintain native-like conditions.
• Mass spectrometer: Agilent 6230C Accurate Mass TOF LC/MS with AJS ion source; tuned for native conditions (e.g., elevated source temperatures, sheath gas flow, and fragmentor/skimmer voltages optimized for intact proteins).
• Software: MassHunter acquisition, BioConfirm, and MassHunter Qualitative Analysis for data processing, deconvolution and proteoform assignment.

Main results and discussion

Key findings from intact native analysis in plasma:

• Direct detection of intact native trastuzumab in neat plasma without affinity enrichment. Native charge-state spectra and deconvoluted masses showed preserved glycoform profiles with excellent mass accuracy (low ppm deviations reported for major glycoforms).
• Analytical sensitivity was demonstrated across serial dilutions from 1 µg/µL down to 62.5 ng/µL, with clear native charge-state signals (e.g., the 27+ charge state) and retained glycoprofile information at low concentration.
• T-DM1 (an ADC) presented greater analytical complexity due to DAR heterogeneity and coeluting endogenous plasma proteins. Extending the 2D IEX gradient alone was insufficient to resolve interferences; however, automated multiple heart-cutting allowed separate high-resolution analysis of SEC fractions, enabling detection and deconvolution of T-DM1 species with different DARs directly from plasma.
• UV-guided heart-cutting simplified method setup and targeted transfer of intact protein zones from SEC to IEX, improving selectivity and preserving native structure for downstream MS analysis.

Benefits and practical applications of the method

The demonstrated workflow offers several practical advantages:

• Non-affinity, native-compatible approach reduces sample manipulation and potential bias introduced by immunocapture, facilitating more complete proteoform profiling.
• Orthogonal 2D separations with precise heart-cutting increase selectivity and mitigate ion suppression from abundant plasma constituents.
• High mass accuracy and sensitivity from the TOF system support confident identification of glycoforms and DAR species, relevant for biopharmaceutical characterization, PK/PD studies, biosimilar assessment, and ADC heterogeneity monitoring.
• Automated, UV-guided method development accelerates deployment for complex biological samples and supports routine workflows in research and QC environments.

Limitations and critical considerations

• Endogenous plasma proteins can still co-elute and interfere, particularly for highly heterogeneous analytes like ADCs; multiple heart-cuts increase analysis time and data complexity.
• Detection limits depend on instrument sensitivity and the efficiency of fraction transfer; further enrichment or orthogonal strategies may be needed for lower-abundance proteoforms in clinical samples.
• Native conditions constrain solvent and buffer choices (volatile salts like ammonium acetate), which may limit certain chromatographic options.

Future trends and potential applications

Anticipated developments and opportunities for this approach:

• Integration with higher-resolution mass analyzers (e.g., Orbitrap-based native MS) and advanced fragmentation (native top-down MS/MS) to yield sequence-level and localization information for PTMs and drug conjugation sites.
• Further automation and intelligent heart-cut algorithms driven by real-time UV/MS feedback to optimize cut timing and reduce analyst input.
• Application to clinical and translational studies for direct plasma proteoform monitoring, biotherapeutic pharmacokinetics, immunogenicity screening, and characterization of complex biologics without dedicated affinity reagents.
• Improved data analysis pipelines for deconvolution, DAR assignment and proteoform annotation to handle multi-cut datasets and heterogeneous species routinely.

Conclusion

The automated 2D-LC/TOF workflow with multiple heart-cutting demonstrates a viable, non-affinity strategy for native intact protein analysis directly in plasma. It achieves high sensitivity and mass accuracy for monoclonal antibodies and, with targeted multi-cutting, can resolve ADC DAR heterogeneity despite plasma interferences. The approach reduces dependence on immunocapture, preserves native proteoform integrity, and is suitable for biopharmaceutical characterization and advanced proteomics applications when combined with careful sample handling and optimized chromatographic transfer.

Reference

1. Fischer MS, et al. Native Top-Down Proteomics of Endogenous Protein Complexes Enabled by Online Two-Dimensional Liquid Chromatography. Analytical Chemistry. 2025.
2. Lambert JM, et al. Ado-trastuzumab Emtansine (T-DM1): An Antibody–Drug Conjugate (ADC) for HER2-Positive Breast Cancer. Journal of Medicinal Chemistry. 2014.