The Role of Multi-Angle Light Scattering in Biosimilar Characterization and Approval

Significance of the topic

The characterization of biotherapeutics and biosimilars is central to regulatory approval, product quality, and patient safety. Multi-angle light scattering (MALS) coupled with liquid chromatography delivers absolute molar mass and size information in solution without calibration standards, enabling reliable quantification of monomers, oligomers, aggregates and fragments in native formulation conditions. This capability makes MALS a core technique in comparability assessments, post-approval process changes, forced-degradation studies and stability testing where accurate mass and aggregation profiles directly inform risk assessment and regulatory decisions.

Objectives and study overview

This document summarizes three published use-cases demonstrating how SEC-MALS and CEX-MALS were applied to support biosimilar comparability and stability evaluations: (1) a post-approval cell line change for a bevacizumab biosimilar (IBI305), (2) a cross-market similarity assessment of a ustekinumab biosimilar candidate (SB17) and (3) comparative forced-degradation and stability analysis of marketed bevacizumab formulations. The underlying objective in all studies was to determine whether process changes or product differences affect key molecular attributes that could impact safety, efficacy or regulatory acceptance.

Methodology and instrumentation

Key methodological features used across the studies:

• Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) with differential refractive index (dRI) and UV detectors for absolute molar mass, monomer/aggregate quantitation and empirical extinction coefficient determination.
• Cation-exchange chromatography coupled to MALS (CEX-MALS) to measure molar mass of separated charge isoforms and gain insight into degradation/aggregation pathways.
• Forced-degradation matrices including thermal (50 ± 2 °C, up to 14 days), chemical (acidic, neutral, basic pH exposures for hours) and mechanical stress (agitation at 200 rpm for up to 24 hours) to probe stability and aggregation propensity.
• Orthogonal physicochemical and functional assays were used in combination with MALS data to support holistic comparability conclusions.

Instrumentation used

• DAWN multi-angle light scattering photometer for absolute molar mass and size determination.
• HPLC/SEC systems (reported integrations included high-performance LC such as Arc Premier) and high-performance SEC columns (MaxPeak Premier) for robust separation.
• Detectors and software: differential refractive index (dRI), UV280, ASTRA and Empower for data acquisition, analysis and compliance-ready data management.

Main results and discussion

• Post-approval cell line change for bevacizumab biosimilar (IBI305): SEC-MALS showed essentially unchanged monomer molar mass and monomer content before and after switching host cell lines, with monomer MW ranges near 155 kDa and monomer content ≈99%. High-molecular-weight species (HMWS) had molar masses approximately 309–327 kDa and HMWS content remained low; in some comparisons the post-change product showed similar or slightly reduced HMWS percentage versus reference. CEX-MALS additionally measured molar masses of separated charge isoforms to evaluate degradation pathways. Overall, MALS data supported that the increased-expression host did not introduce meaningful molecular deviations that would preclude re-approval.
• Similarity assessment for SB17 (ustekinumab biosimilar): SEC-MALS combined with dRI and UV280 quantified main peak, HMWS and low-molecular-weight species and enabled empirical determination of extinction coefficients by integrating dRI and UV signals. Absolute molar mass measurements from SEC-MALS demonstrated close agreement between SB17 and both US and EU reference lots, strengthening the comparability package ultimately leading to regulatory approval.
• Comparative stability and forced-degradation of bevacizumab formulations: SEC-MALS tracked aggregation under thermal, chemical and mechanical stress. Short thermal exposure (up to 1 day at 50 °C) preserved stability, whereas longer exposure (up to 14 days) produced progressive accumulation of dimers and higher oligomers. Mechanical agitation promoted marked aggregation and conformational changes within 24 hours across products, suggesting formulation surfactant (e.g., polysorbate 20) and handling are critical contributors to agitation-induced instability. These findings provide a molecular basis for observed stability differences and inform formulation and handling controls.

Benefits and practical applications of the method

• Absolute molar mass without calibration eliminates biases introduced by column variability and retention-time based methods, improving comparability confidence across labs and regulatory submissions.
• The ability to measure in formulation buffer (native conditions) offers realistic assessment of product behavior and aggregation propensity relevant to storage and administration.
• MALS supports a broad set of analytical questions: monomer/aggregate quantitation, oligomer identification, molar mass of charge isoforms, empirical extinction coefficient determination and monitoring of forced-degradation effects.
• Integration with standard HPLC/SEC platforms and compliance-ready software facilitates routine use in QC, comparability studies and regulatory dossiers.

Future trends and potential applications

• Broader coupling of MALS with orthogonal separation modes (e.g., ion-exchange, hydrophobic interaction, AF4) and with mass spectrometry to merge absolute mass, detailed structural and sequence-level information.
• Increased automation and high-throughput SEC-MALS workflows for comparability screening across multiple lots, formulations and stress conditions.
• Advances in detector sensitivity and expanded angular range to improve characterization of very large assemblies, heterogeneous conjugates and nanoparticle-based therapeutics (e.g., ADCs, VLPs, lipid nanoparticles).
• Integration of MALS datasets into centralized data management and machine-learning platforms to detect subtle pattern differences across manufacturing changes and to predict stability outcomes.
• Regulatory standardization of MALS-derived quality metrics as part of accepted comparability toolboxes for biosimilar and post-approval change submissions.

Conclusion

SEC-MALS and CEX-MALS are powerful, practical tools for the rigorous physicochemical characterization required in biosimilar development, post-approval process changes and stability testing. The highlighted studies illustrate how absolute molar mass data, aggregation profiling and extinction coefficient determination strengthen comparability claims and guide risk-based decisions on manufacturing or formulation changes. When combined with orthogonal assays and robust data management, MALS substantially improves the analytical foundation for regulatory submissions and product lifecycle control.

References

1. Wu Z., et al. Comparability strategy and demonstration for post-approval production cell line change of a bevacizumab biosimilar IBI305. Antibody Therapeutics. 2023;6(3):194–210.
2. Yang S.Y., et al. Characterization for the similarity assessment between the proposed biosimilar SB17 and ustekinumab reference product using state-of-the-art analytical methods. Analytical Methods/Drugs R D. 2025.
3. Bana A.A., et al. Comparative stability study and aggregate analysis of bevacizumab marketed formulations using advanced analytical techniques. Heliyon. 2023;9(9):e19478.
4. ICH Q5C, Q6B, Q1B framework referenced for stability testing and characterization of biotechnological products.