NATIVE MASS SPECTROMETRY DISTINGUISHES HOMO VS HETERO NON-COVALENT DIMERIC AGGREGATES OF MONOCLONAL ANTIBODIES

Importance of the Topic

Monoclonal antibody (mAb) therapeutics can form non-covalent aggregates that compromise efficacy, safety and shelf-life.
Distinguishing between homodimeric and heterodimeric species is critical for quality control, formulation development and understanding degradation pathways.
Native mass spectrometry offers a gentle, high-resolution approach to characterize intact soluble mAb aggregates.

Aims and Study Overview

The study aimed to establish an assay that combines size-exclusion chromatography (SEC) and native nano-electrospray ionization mass spectrometry (nano-ESI-MS) to:

• Isolate and enrich monomeric and high-molecular-weight (HMW) fractions of two stressed Bristol-Myers Squibb mAbs (mAb1 and mAb4).
• Detect and assign monomers, homodimers, heterodimers, trimers and tetramers under native conditions.
• Delineate homo- vs hetero-dimer formation in a mixed stressed mAb1+mAb4 sample.

Methodology

Sample Preparation and Separation:

• Exchange mAb formulations into 100 mM ammonium acetate, pH 6.5, using spin-column SEC.
• Perform preparative SEC-UV to collect enriched monomer and HMW aggregate fractions from individually stressed and mixed samples (40 °C, 12 weeks).
• Validate purity and monomer:HMW ratios via analytical SEC combined with multi-angle light scattering (SEC-MALS).

Instrumentation

• Synapt G2-Si quadrupole time-of-flight (QTof) mass spectrometer with nanoESI source.
• Micro Bio-Spin P6 columns for buffer exchange.
• Preparative and analytical HPLC systems for SEC-UV and SEC-MALS separations.

Main Results and Discussion

• Native ESI-MS spectra revealed distinct charge-state distributions for monomer (~150 kDa), homodimers (~300 kDa), heterodimers (~297 kDa), trimers (~445 kDa) and tetramers (~595 kDa).
• SEC enrichment was essential to detect low-abundance HMW species in stressed samples.
• MaxEnt1 deconvolution provided accurate mass assignments that closely matched theoretical values for AA, BB and AB dimer types.
• Control experiments confirmed the absence of artifactual dimer formation during ESI when mAb1 and mAb4 were mixed online.

Benefits and Practical Applications

• Enables rapid, label-free assessment of non-covalent mAb aggregates under native conditions.
• Provides direct molecular weight confirmation of HMW species, improving analytical rigor in biologics development.
• Supports comparability studies for biosimilars and formulation screening by resolving homo- vs hetero-aggregate populations.

Future Trends and Opportunities

• Integration of online SEC-MS workflows for real-time aggregate profiling during manufacturing and formulation optimization.
• Advancements in high-resolution native MS instrumentation to resolve larger complexes and subtle structural variants.
• Application of data-driven and AI-guided tools to predict aggregation hotspots and guide protein engineering for enhanced stability.
• Expansion to multi-specific and bispecific antibody platforms, vaccine constructs and other complex biotherapeutics.

Conclusion

Native nano-ESI mass spectrometry coupled with preparative SEC effectively distinguishes homodimeric and heterodimeric mAb aggregates.
This approach delivers precise mass assignments for non-covalent HMW species, enhancing the analytical toolkit for therapeutic antibody characterization.

References