Bispecific & Multispecific Antibody Characterization
Multispecific and bispecific antibodies (MsAbs / bsAbs) combine two or more antigen-binding specificities within a single molecule, enabling advanced mechanisms such as T-cell engagement, dual checkpoint blockade or receptor clustering. The wide variety of formats (e.g. IgG-like, Fc-less, knob-into-hole, CrossMab, tandem scFv) makes evaluation and characterization of critical quality attributes (CQAs) particularly sensitive and complex. Correct chain pairing, stoichiometry, format integrity and balanced binding to all targets are essential for efficacy, safety and manufacturability, and require dedicated multispecific antibody characterization strategies.
At Biofidus, we offer a broad, state-of-the-art analytical platform for multispecific and bispecific antibodies to support innovator biologics development as well as biosimilar comparability studies.
We cover structural and functional characterization, including in-depth primary structure and sequence confirmation, amino acid and post-translational modifications, impurities such as homodimers or mispaired species, and target-relevant functionality in cell-based assays.
We are specialists in antibody glycosylation analysis, a key modification with strong impact on function, stability and immunogenicity of biopharmaceutical molecules. Our extensive experience allows us to guide you through the available methods and define the optimal, ICH-aligned analytical strategy for your MsAb / bsAb project.
Multispecific Antibody CQAs and Analytical Techniques
| Critical Quality Attribute | Biofidus Techniques | Development Purpose |
|---|---|---|
| Chain pairing & format integrity | Intact/subunit LC-MS, peptide mapping LC-MS/MS, CE-SDS, SEC | Correct pairing, homodimers, mispaired species |
| Identity & primary structure | Intact/subunit LC-MS, peptide mapping LC-MS/MS | Identity, sequence, N- and C-terminus |
| Post-translational modifications | Peptide mapping LC-MS/MS, intact/subunit LC-MS | Oxidation, deamidation, isomerization, glycation, clipping |
| Glycosylation | HILIC-FLD-MS, glycopeptide LC-MS/MS, HPAEC-PAD | Glycan profile, occupancy, monosaccharides, sialic acids |
| Disulfide bonds & free thiols | Non-reduced LC-MS/MS, free-thiol assay, non-reducing CE-SDS | Disulfide pairing and scrambling, free cysteines |
| Higher-order structure | CD, FTIR, intrinsic fluorescence | Secondary/tertiary structure |
| Thermal stability | DSC, nanoDSF, DLS | Unfolding, Tm, aggregation onset |
| Size variants & aggregation | SEC-UV/MALS, AUC, DLS | Monomer, aggregates, molecular size |
| Fragments & purity | Reducing/non-reducing CE-SDS, SEC-UV/MALS | Fragments, clipping, purity |
| Particles | Light obscuration, MFI, DLS | Subvisible particles |
| Charge variants | IEX (CEX and AEX), cIEF, CZE | Acidic/basic variants |
| Hydrophobicity variants | HIC, RP-HPLC | Hydrophobic and hydrophilic variants |
| Product-related impurities | SEC, CE-SDS, IEX/cIEF, LC-MS/MS | Aggregates, fragments, modified forms |
| Process-related impurities | HCP ELISA/LC-MS, qPCR, Protein A ELISA | HCP, residual DNA, Protein A |
| Target binding | SPR, ELISA, flow cytometry | Affinity, kinetics, specificity, cellular binding |
| Fc-mediated binding | SPR, ELISA | FcγR, FcRn, C1q binding |
| Biological activity | Cell-based assays including reporter gene assays, signal transduction, viability and cytotoxicity (e.g., ADC cytotoxicity) | Potency, mechanism of action |
| Effector function | ADCC (using reporter or primary cells as effector cells), CDC, ADCP | Fc-mediated activity |
| Internalization | Flow cytometry-based internalization analysis | Internalization kinetics |
| Protein concentration | UV A280, amino-acid analysis | Concentration, extinction coefficient |
| Stability & degradation | Forced degradation + stability-indicating methods | Degradation pathways, shelf life |
| Comparability & biosimilarity | Orthogonal structural and functional analytics | Batch, process, reference comparison |
We adapt our platform methods to the properties of your drug substance, its individual formulation matrix, and your specific analytical question, and qualify them as needed according to ICH guidelines.
Get in contact with us to quickly find the optimal setup for your analytical challenge and to speak directly from expert to expert.
