Titer and charge-based heterogeneity multiattributemonitoring of mAbsin cell culture harvest using 2D ProACEX MS

Significance of the Topic

Monoclonal antibodies (mAbs) require rigorous monitoring of both quantity (titer) and quality attributes such as charge variants to ensure safety and efficacy. Traditional workflows involve separate Protein A (ProA) affinity capture for titer followed by cation-exchange chromatography (CEX) for charge heterogeneity, consuming significant time and resources. Integrating these assessments into a single, rapid two-dimensional liquid chromatography–mass spectrometry (2D-LC–MS) workflow addresses key industry demands for speed, accuracy, and streamlined analytics in biopharmaceutical development.

Objectives and Study Overview

• Develop a native 2D-LC–MS method combining ProA capture and CEX separation to quantify mAb titer and charge variants in a single run.
• Validate method performance against conventional non-volatile buffer protocols for speed, resolution, and accuracy.
• Demonstrate applicability to trastuzumab cell culture harvest and its biosimilars for biosimilarity assessment.

Methodology

The workflow employed a dual‐dimension LC system:

• First Dimension (1D): Protein A chromatography with volatile mobile phases, 2.5-minute linear gradient, 1.0 mL/min flow, capturing mAb from clarified cell culture harvest.
• Interface: Multiple heart-cutting valves and active solvent modulation to transfer the ProA elution peak directly into the second dimension.
• Second Dimension (2D): CEX separation on a short SCX column using volatile buffers, 5-minute linear gradient, 1.0 mL/min flow, resolving acidic and basic variants.
• MS Detection: Coupled to a Q-TOF system under native conditions for intact mass determination of each charge species.
• Linearity Study: Injection amounts from 5 µg to 160 µg trastuzumab to assess quantitative response (R² ≥ 0.9998).

Instrumentation Used

• Agilent 1290 Infinity II 2D-LC system with ProA and CEX columns.
• Multiple Heart-Cutting Valves and ASM capillary interface.
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer.

Main Results and Discussion

• The 2D ProA-CEX-MS method achieved complete separation of six charge variants in 15 minutes, matching or exceeding resolution of traditional workflows.
• Intact mass deconvolution identified major acidic (deamidation) and basic (isomerization, succinimide) species for trastuzumab reference and four biosimilars.
• Titer quantitation exhibited excellent linearity (R² = 0.9999) across the tested range, demonstrating robust sensitivity.
• Overall runtime reduced from ~60–90 minutes (separate ProA and CEX with buffer exchanges) to 8 minutes for titer alone and 15 minutes including MS characterization.

Practical Benefits and Applications

• Single-workflow solution eliminates offline fraction collection and desalting steps.
• Rapid throughput supports high-throughput process development and in-line quality control.
• Enhanced analytical agility for biosimilarity assessments and comparability studies.

Future Trends and Applications

• Expansion to additional critical quality attributes (glycoforms, oxidation) via third-dimension or parallel detection.
• Integration with online process analytical technology (PAT) platforms for real-time monitoring.
• Application to next-generation biotherapeutics (bispecifics, ADCs) requiring complex heterogeneity profiling.

Conclusion

The native 2D ProA-CEX-MS approach offers a unified, time-efficient method to quantify mAb titer and resolve charge variants in a single 15-minute run. Demonstrated linearity, resolution, and compatibility with biosimilarity studies highlight its potential as a next-generation analytical platform for biopharmaceutical development.

Reference

• Murisier A. et al. J. Chromatogr. A 1655 (2021) 462499.
• Rogers R.S. et al. mAbs 7 (2015) 881–890.
• Rathore A.S., Sarin D. Trends Biotechnol. (2023) 1–11.
• Sarin D. et al. Talanta 276 (2024) 126232.