5 Analytical Challenges in Biosimilar Characterization and Comparability

• Biosimilar characterization is the scientific foundation of biosimilar development and regulatory approval. In 2026, analytical capabilities in recombinant protein analytics have reached extraordinary sensitivity and resolution, yet the demonstration of drug similarity remains constrained by biological variability, analytical limitations, and model-dependent interpretation. 
• Modern biosimilarity studies require orthogonal method strategies, rigorous qualification of biological assays, and transparent documentation of residual uncertainty. 

Glycosylation Comparability and Profiling 

Glycosylation comparability remains one of the most technically demanding elements of biologics characterization. Glycosylation is highly sensitive to cell line, media composition, culture conditions, and purification steps, making biosimilar process development intrinsically linked to glycan outcome. For monoclonal antibodies, Fc-fusion proteins, and other biologics, glycan structures influence Fc receptor binding, complement activation, serum half-life, and potentially immunogenicity.¹

In the analysis of therapeutic proteins, one of the main challenges in N-glycan characterization is that the choice of analytical strategy strongly depends on the objective of the study and on a clearly defined research question. Precise and accurate determination of the relative abundance of individual glycoforms requires a different approach than the unambiguous identification of glycan structures or their assignment to specific glycosylation sites. Analysis of released glycans, for example by HILIC-FLD following PNGase F-mediated release of N-glycans and fluorescent labeling, provides high repeatability, accuracy, and precision for relative quantification of individual glycoforms. However, this approach has limitations for proteins containing multiple glycosylation sites, since it does not provide site-specific information. It may also be insufficient when glycans co-elute or when more in-depth glycan identification is required. In contrast, mass spectrometry-based techniques, such as HILIC-MS and RP-MS/MS enable structural identification of glycans and significantly expand the qualitative scope of the analysis. Glycopeptides analysis by RP-MS/MS is particularly important when a protein contains multiple glycosylation sites, because it allows assignment of specific glycan structures to particular sites within the protein. However, MS-based methods are often less reliable for relative abundance measurements, as the glycosylation can substantially affect ionization efficiency. Therefore, in practice, the main challenge lies not in selecting a single “best” method, but in combining complementary analytical approaches appropriately. HILIC-FLD is highly effective for precise and accurate relative quantification of glycoforms, MS enables their structural identification, and glycopeptide analysis is essential when site-specific glycosylation information must be retained.^2-5

The analytical challenge for a biosimilar CDMO lies in distinguishing clinically irrelevant microheterogeneity from meaningful shifts in Fc-mediated function. Justification requires integrated analysis combining glycosylation comparability, Fc receptor binding kinetics, and functional bioassay outcomes. The combination of orthogonal analytical methods increases the reliability of glycan characterization. Regulatory expectations in biosimilar testing require not only qualitative similarity in glycan species but also quantitative comparability within justified ranges, making glycosylation profiling central to biosimilar characterization. Proper documentation in biosimilarity studies must therefore clearly link structural glycosylation profiling to functional equivalence.⁶

Host Cell Proteins and Process-Related Impurity Profiling

Residual host cell proteins (HCPs) represent one of the most persistent blind spots in biosimilar characterization. Although ELISA-based HCP quantification remains the regulatory gold standard, it is fundamentally limited by antibody reagent coverage. No polyclonal antibody reagent can theoretically recognize all process-specific HCP species present in biosimilar production.^7,8

Orthogonal LC-MS-based proteomics can identify individual host cell proteins species, but quantitative reproducibility remains limited due to matrix complexity, ionization variability, and absence of universal reference standards. Unlike physicochemical assays, there is no globally harmonized quantitative acceptance criterion for individual HCP species in biosimilar assessment.⁹

For this reason, ELISA and LC-MS should be viewed as complementary rather than competing approaches in HCP assessment. ELISA provides highly sensitive measurement of total HCP content and remains well suited for routine quality control and  process monitoring. In contrast, LC-MS enables characterization of the HCP profile at the level of individual protein species, supporting risk assessment. When applied together, these orthogonal methods reduce analytical uncertainty and provide a more complete understanding of residual process-related impurities. Therefore, whenever feasible, the combined use of ELISA and MS strengthens HCP control strategies in biosimilar development and supports a more scientifically justified assessment of product quality. 

Host Cell Protein ELISA Assay Qualification and Regulatory Justification

Method qualification for HCP ELISA requires demonstrating reagent coverage against process-specific HCP populations, but such validation often relies on spiking or mock harvest preparations that do not fully reflect downstream clearance patterns. In biosimilar comparability studies, differences in HCP profiles between originator and biosimilar may reflect distinct manufacturing biology rather than safety risk.⁷

The key biopharma challenge is to construct a risk-based impurity profile justification supported by orthogonal bioanalytics, clearance validation data, and toxicological rationale. Transparent discussion of analytical detection limits and blind spots is increasingly expected in regulatory submissions for biosimilar medicines.

In 2020, United States Pharmacopeia (USP) convened an expert panel of industry, academic, and regulatory specialists to address the best practices for HCP analysis by MS. This endeavor resulted in the release of General Chapter <1132.1> “Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry” in December 2024, which will become official on May 1, 2025. In the sections below, we provide a brief review of standardized techniques used in MS-based HCP analyses.

Potency and Functional Assay Panel with High Intrinsic Variability

Functional assays translate structural attributes into biological effect and are therefore central to biosimilar characterization. However, potency biologic assays are inherently variable and difficult to validate to the precision levels expected in small-molecule analytics.

Cell line heterogeneity, receptor density fluctuations, passage number effects, and environmental conditions introduce biological noise that cannot be eliminated, only controlled. Even well-optimized functional bioassays may exhibit relative standard deviations of 10–20% or higher, complicating equivalence margin calculations.¹⁰

Assay format differences between originator historical methods and biosimilar sponsor systems further complicate drug similarity comparisons. In some cases, reference product drift over time introduces additional variability independent of biosimilar development.

Validation of potency cell-based assays requires demonstration of accuracy, precision, linearity, and robustness, yet biological systems rarely conform to ideal validation models. Parallel-line analysis assumes comparable dose-response curve shapes, but subtle slope differences may reflect assay noise rather than true product differences.¹¹

Target and Fc Receptor Binding Kinetics and Avidity

Binding kinetics assessment using surface plasmon resonance (SPR) is a cornerstone of recombinant protein analytics. This technology provide real-time measurements of association and dissociation rates, yet their outputs are highly dependent on experimental configuration and kinetic modeling assumptions.¹²

Fcγ receptor binding is highly sensitive to glycosylation microheterogeneity, making analytical variability inseparable from structural variation. Additionally, recombinant receptor constructs used in vitro may not fully reflect native receptor clustering or membrane context.¹³

In comparability studies, the challenge lies in demonstrating that binding similarity across multiple receptor subtypes supports overall drug similarity. Orthogonal approaches, including cell-based ADCC functional assays, are often required to contextualize kinetic differences.¹⁴

Model Dependence and Experimental Artifacts

Selection of a 1:1 Langmuir model, heterogeneous ligand model, or bivalent analyte model can significantly alter calculated affinity constants. For monoclonal antibodies, avidity effects often violate simple binding assumptions, particularly when antigen density on sensor surfaces is high.¹⁵

Mass transport limitation, rebinding artifacts, and ligand immobilization chemistry can introduce systematic bias. Even when biosimilar characterization data appear comparable numerically, differing model assumptions may obscure underlying differences or exaggerate insignificant ones.

Stability and Degradation Pathway Comparability Under Real-World Handling 

Biosimilar stability assessment extends beyond ICH long-term and accelerated conditions. Transport excursions, agitation, freeze-thaw cycles, and light exposure may reveal degradation pathways not fully captured in controlled studies.^6,16

Two products may share qualitatively similar degradation pathways while differing quantitatively in degradation kinetics. Determining whether such differences are clinically meaningful requires integration of structural, functional bioassay, and potency bioassay data.

An additional and often underappreciated dimension of stability comparability is the role of formulation composition. Excipients such as buffers, sugars, amino acids, surfactants, and stabilizers directly influence protein conformational stability, aggregation propensity, oxidation susceptibility, and surface adsorption behavior. Even when the active substance is highly similar, differences in formulation can lead to divergent degradation kinetics or altered dominant degradation pathways. Formulation also governs the product’s resilience to external stressors. Protein–excipient interactions may either mitigate or exacerbate structural perturbations induced by temperature fluctuations or mechanical stress. In some cases, equivalent degradation pathways are observed qualitatively, but formulation-dependent microenvironmental effects result in different rates or extents of degradation under identical stress conditions.

Conclusion

Biosimilar characterization in 2026 is defined not by lack of analytical technology, but by the inherent biological complexity of biosimilar medicines.¹⁷ Gold-standard methods in bioanalytics remain indispensable, yet none are perfect. Each method introduces interpretational constraints, validation challenges, and statistical complexity that must be transparently managed in comparability studies. To be sure of the analytical results, it is recommended to verify the tested parameter using at least two orthogonal methods.

FAQ

Prepared by:

Małgorzata Urbaniak

Manager of Analytical Methods (Physicochemical Methods)

m.urbaniak@mabion.eu

Małgorzata Urbaniak

Manager of Analytical Methods (Physicochemical Methods)

Małgorzata Urbaniak has been working at Mabion since 2015. She began her career at the company as a Junior Biochemist Specialist in the Research and Development Department. Since then, now as a Senior Biochemist Specialist, she has been involved in developing analytical methods for assessing the quality characteristics of therapeutic proteins. She is particularly fond of methods based on high-performance liquid chromatography coupled with mass spectrometry.

Jakub Knurek

Marketing Specialist

j.knurek@mabion.eu

Jakub Knurek

Marketing Specialist

Jakub Knurek is a marketing and business development expert specializing in CDMO operations with scientific and biopharmaceutical background. During the COVID-19 pandemic, he contributed to the development of the Novavax protein vaccine. As a side project, he runs his own pharma company.

As the author of numerous articles, he is recognized as an important voice of the younger generation of scientists. He is a member of Mabion’s representation at industry trade shows, including BIO International Convention 2025 in Boston, 2nd Global Biologics India 2025 in Hydrabad, CPHI 2024 in Milan, CPHI 2023 in Barcelona, Life Sciences Baltics 2023 in Vilnius. He actively participates in academic, business and industry discussions, such as the “Building the Future Pharma Workforce” panel at CPHI Milan 2024.

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