Bispecific antibodies are an increasingly important class of therapeutics, providing the ability to bind two distinct targets and enabling novel mechanisms of action. However, their structural complexity introduces analytical challenges, particularly when characterizing critical quality attributes (CQAs) such as charge heterogeneity and size variants.
DDN spoke with Srinivasa Rao, Principal Scientist at Bio-Techne, and Nina Jajčanin Jozić, Senior Applications Scientist at Refeyn, to explore a newly developed workflow that combines imaged capillary isoelectric focusing (icIEF) fractionation with mass photometry (MP) to address these challenges in a streamlined manner.
Why is characterization of bispecific antibodies particularly challenging?
Bispecific antibodies must maintain correct pairing of their constituent chains to ensure safety and efficacy. Even small deviations in structure, such as mispaired chains or partial assemblies can affect therapeutic performance.
Traditionally, scientists rely on separate techniques to analyze different CQAs. Ion-exchange chromatography is used to assess charge variants, while size-exclusion chromatography (SEC) evaluates size heterogeneity. However, these approaches are difficult to integrate into a single workflow because SEC often requires larger sample volumes and buffer exchange steps that are incompatible with the small fractions generated during charge variant separation.
This disconnect leaves charge and size attributes largely uncorrelated, limiting insight in bispecific antibody analysis.
How are you addressing these workflow inefficiencies?
We have designed a new workflow that combines icIEF fractionation, performed on the MauriceFlex platform, with MP analysis using the TwoMP system.
In the first step, icIEF fractionation separates antibodies into distinct charge variant fractions (acidic, main, and basic species) with high resolution, enabling collection at over 70 percent purity. These fractions can then be analyzed directly by MP, without the need for buffer exchange. MP operates at the single molecule level to determine molecular mass and quantify size variants, including fragments and aggregates.
By integrating these two techniques, the workflow enables simultaneous characterization of charge and size variants in a single pipeline, overcoming limitations of traditional methods.
What are the key advantages of combining icIEF with MP?
Each technique contributes complementary strengths:
- icIEF fractionation provides high-resolution separation of charge isoforms and enables isolation of distinct antibody populations.
- MP detects and quantifies size variants, including low-abundance fragments and aggregates that may be difficult to observe with SEC.
Importantly, the combined workflow is both fast and efficient. The full process can be completed in approximately four hours and requires only 1–5µL of sample per fraction, minimizing sample consumption while reducing hands-on effort.
This integration provides a more comprehensive understanding of antibody quality attributes while simplifying experimental workflows.
How was the workflow applied in the case study?
The workflow was applied to compare an innovator bispecific antibody, mosunetuzumab (MOS), with a research-grade biosimilar.
Samples were analyzed under three conditions:
- Native (control)
- Heat stress (45°C for seven days)
- Chemically induced aggregation using a cross-linker, BS3
This experimental design allowed researchers to evaluate how each molecule responds to stress and how charge and size variants evolve under different conditions.
What did icIEF reveal about charge heterogeneity?
icIEF analysis highlighted distinct differences between the innovator and biosimilar antibodies.
The innovator showed a dominant main peak with relatively minor acidic and basic variants across all conditions. In contrast, the biosimilar exhibited an additional basic peak, even under native conditions, which became more pronounced under stress.
Quantitative analysis confirmed that the biosimilar had a higher relative abundance of basic variants, indicating greater charge heterogeneity compared with the innovator molecule.
How did MP enhance the analysis?
MP provided deeper insight into the size variations associated with these charge differences.
In the innovator samples, MP analysis detected a dominant species corresponding to the expected full antibody mass (~150kDa), with minimal impurities.
However, the biosimilar samples revealed additional species, including ~75kDa fragments (half-antibodies) and ~225kDa species (1.5-antibody forms), particularly enriched in the basic fractions.
These findings suggest assembly-related inconsistencies in the biosimilar.
What insights were gained under stress conditions?
Stress testing further differentiated the stability of the two molecules.
The innovator antibody demonstrated relatively stable profiles, with only modest increases in low-molecular-weight species and high-molecular-weight species.
In contrast, the biosimilar showed significantly greater variability, with higher levels of fragmentation and aggregation in both the control state and under stress. Certain charge variants were more prone to degradation, highlighting specific regions of vulnerability within the molecule.
Additionally, MP resolved distinct aggregate species, revealing a broader distribution of assemblies in the biosimilar — from partial antibodies to higher-order aggregates — compared to the more stable innovator molecule.
What are the key takeaways of your recent collaboration?
This combined icIEF and MP workflow closes a critical gap in bispecific antibody characterization by enabling direct correlation between charge and size variants within a single analytical pipeline.
The workflow offers a powerful, streamlined platform solution for bispecific antibodies analysis that:
- Enables simultaneous characterization of charge and size variants in less than half a day without buffer exchange nor method development steps.
- Provides quantitative insights into antibody assembly, fragmentation, and aggregation.
- Reveals differences in stability and heterogeneity for biosimilar antibodies development.
The ability to rapidly detect assembly-related impurities and stress-induced changes supports earlier decision-making and potentially accelerates development timelines. Ultimately, this approach delivers a more comprehensive and efficient way to evaluate complex biologics, supporting the development of safer and more effective therapies.
