Determination of Multiple Attributes of Monoclonal Antibodies

Importance of the Topic

Therapeutic monoclonal antibodies are structurally complex molecules with numerous variants arising from size heterogeneity, charge heterogeneity, glycosylation, and post-translational modifications. Comprehensive characterization of these variants directly from cell culture supernatants is critical for biopharmaceutical research, clone selection, process development, and quality control. Multi-attribute analysis that integrates purification, quantitation, and variant profiling in a single automated workflow can accelerate decision making and reduce analytical workload.

Objectives and Study Overview

– Develop a three-dimensional liquid chromatography mass spectrometry platform that combines Protein A affinity purification, a second-dimension multi-method separation (SEC, CEX, HIC), and a third-dimension desalting SEC-MS measurement.
– Assess mAb titer, size variants, charge variants, hydrophobic variants, molecular weight, amino acid sequence information, and post-translational modifications simultaneously and in parallel.
– Demonstrate automated switching between chromatography modes without loss of resolution.
– Evaluate the workflow using commercial trastuzumab and five trastuzumab-producing CHO clones.

Methodology

Sample supernatants were clarified by centrifugation and directly loaded onto a Protein A affinity column for mAb capture. Elution at low pH provided titer determination by UV absorbance at 280 nm. The eluted antibody was loop-collected and heart-cut into a second dimension, where a selector valve directed the sample to one of three modes: size-exclusion chromatography (SEC) for aggregate and fragment analysis, cation-exchange chromatography (CEX) for charge variant profiling, or hydrophobic interaction chromatography (HIC) for hydrophobic variant separation. A final desalting SEC step removed nonvolatile buffers before electrospray ionization mass spectrometry. Automated solvent preconditioning and blank injections ensured compatibility across modes.

Instrumentation

• Agilent InfinityLab 2D-LC System with multiple heart-cutting option, solvent selector and column selector valves
• Agilent 1260 Infinity II Quaternary Pump and 1290 Infinity II Multisampler with column thermostat and diode array detection
• Agilent 6530 LC/Q-TOF with Jet Stream ESI source
• Protein A monolith column, AdvanceBio SEC columns for desalting and size-exclusion, Bio MAb NP5 cation exchange column, AdvanceBio HIC column

Main Results and Discussion

– Titer determination achieved a linear calibration over 0.02 to 2.0 µg/µL with R2 of 0.9998. Five CHO clones showed titers ranging from 0.09 to 1.0 µg/µL.
– SEC profiles revealed differences in high molecular weight and low molecular weight species between the originator and clones, with CHO clones showing increased aggregates and fragments.
– CEX separation identified acidic and basic variants; deconvoluted MS spectra confirmed single and double deamidation, isoaspartate formation, C-terminal lysine truncation, and nonmodified main species.
– HIC separated hydrophobic variants; MS spectra exhibited glycoform distribution and sulfate adduct formation under high salt conditions.
– Deconvoluted SEC-MS enabled identification of covalent and noncovalent dimers in the originator sample and monomeric species in clones.
– Automated switching between SEC, CEX, and HIC modes delivered consistent chromatographic quality without carryover or loss of resolution.

Benefits and Practical Applications

• Integrated purification and multi-attribute analysis reduces total analysis time and manual handling.
• Direct analysis from culture supernatant accelerates clone screening and process optimization.
• Automated 2D mode switching enhances throughput and flexibility for different analytical needs.
• Comprehensive variant profiling (size, charge, hydrophobicity, molecular weight, glycosylation) supports in-depth product characterization and comparability studies.

Future Trends and Applications

The workflow can be extended by exploring alternative buffer systems to improve MS sensitivity in HIC mode and by increasing heart-cut loop capacities for trace variants. Integration with online peptide mapping or fragmentation techniques could further reveal sequence variants and localize modifications. Advances in data processing and machine learning-based deconvolution may enhance variant detection and quantitation. The platform is poised for adoption in high-throughput biopharma screening, comparability studies, and real-time process monitoring.

Conclusion

The presented 3D-LC/MS with 2D multimethod option offers a fully automated and robust multi-attribute analyzer for monoclonal antibodies. It combines Protein A capture, switchable second-dimension separations (SEC, CEX, HIC), and desalting SEC-MS to deliver rapid and comprehensive characterization directly from cell culture supernatants. The approach enhances throughput, flexibility, and variant coverage, making it a valuable tool for biopharmaceutical research and quality control.

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