Analysis of bispecific and multispecific antibody therapeutics using native mass spectrometry on a modified hybrid Orbitrap mass spectrometer

Importance of the Topic

Bispecific and multispecific antibodies represent a rapidly emerging class of therapeutics with the ability to engage multiple targets simultaneously. Their structural complexity, including varied heavy and light chain assemblies and engineered domains, poses significant analytical challenges. Native mass spectrometry coupled with online buffer exchange preserves noncovalent interactions and higher order structures, making it a critical tool for detailed characterization and quality control of these advanced biologics.

Objectives and Study Overview

This study aimed to evaluate the performance of a modified Orbitrap Excedion Pro BioPharma mass spectrometer equipped with a NativePac OBE-1 SEC column for native MS analysis of five bispecific antibodies spanning 127 to 240 kDa. Key goals included high-accuracy proteoform mapping, assessment of glycoform heterogeneity, monitoring of heat-induced aggregation, and investigation of antigen binding under native conditions.

Methodology and Instrumentation

Research-grade bsAb samples (cadonilimab, glofitamab, bafisontamab, zanidantamab, tebotelimab) were diluted to 0.5 mg/mL and introduced via online buffer exchange into 50 mM ammonium acetate. Separation was performed on a NativePac OBE-1 SEC column (2.1×50 mm) at 0.2 mL/min, 25 °C. A Thermo Scientific Orbitrap Excedion Pro BioPharma hybrid mass spectrometer operated in intact protein mode with a mass range up to m/z 12,000. Source settings included 3.8 kV spray voltage, 275 °C transfer temperature, and 130 eV in-source CID. Data acquisition used Xcalibur 4.7 and deconvolution employed the ReSpect algorithm in BioPharma Finder 5.3.

Main Results and Discussion

All five bsAbs were confidently characterized with mass accuracy better than 10 ppm for the most abundant proteoforms. Cadonilimab showed clear resolution of multiple N-glycoforms around the +31 charge state. Bafisontamab (~236–241 kDa) exhibited five major glycoforms, including immature and truncated N-glycans. Heat stress of tebotelimab at 37 °C for 72 h led to dimer formation (~1.4% relative), detected by a distinct dimeric charge envelope. Incubation with PD-1 and LAG-3 antigens did not yield stable complexes but induced detectable shifts in the charge distribution, indicating conformational changes.

Benefits and Practical Applications

• High-throughput, label-free analysis preserving native structure
• Accurate proteoform and glycoform profiling with mass errors below 10 ppm
• Detection of aggregation and conformational changes under stress or ligand binding
• Applicability to quality control, stability testing, and structure–function studies

Future Trends and Applications

Extending native MS to larger multispecific constructs beyond 250 kDa through improved ion optics and extended mass range. Integration of ion mobility for conformational and collision cross-section analysis. Automation of online buffer exchange workflows and AI-assisted deconvolution for increased throughput. Coupling with orthogonal techniques such as HDX-MS and SEC-MALS for comprehensive structural characterization.

Conclusion

Online buffer exchange native MS on a modified Orbitrap Excedion Pro BioPharma platform offers a robust, accurate method for detailed analysis of bispecific and multispecific antibody therapeutics. The approach enables high-resolution mapping of proteoforms and glycoforms, monitoring of aggregation and conformational dynamics, and evaluation of antigen binding in a single workflow. This methodology supports advanced research and development, formulation screening, and quality control in next-generation antibody development.

References

• Li H, Er Saw P, Song E. Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020;17:451–461.
• Zhu L, Glick J, Flarakos J. Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics. AAPS J. 2020;22:130.
• Liu W, Jayasekera HS, Sanders JD, Zhang G, Viner R, Marty MT. Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry. Anal Chem. 2023;95(47):17212–17219.