Antibody Characterization
As major components of humoral immunity, IgG antibodies and other therapeutic proteins have been at the centre of biologic drug development for decades. These molecules are highly specific and structurally complex, so protein and antibody characterization require coordinated, multi-dimensional analytical workflows rather than a single assay.
At Biofidus, detailed analysis and characterization of therapeutic proteins and antibodies is part of our daily routine. Our portfolio spans in-depth primary structure analysis and sequence confirmation, amino acid and post-translational modifications (PTMs), glycosylation profiling, and binding and functionality testing in cell-based assays. We are specialists in antibody glycosylation analysis, one of the most critical protein modifications influencing function, stability and immunogenicity of biopharmaceuticals. Using advanced LC-MS/MS, chromatographic, electrophoretic and bioassay platforms, we design fit-for-purpose protein characterization packages for monoclonal antibodies, bispecific antibodies, fusion proteins and other biologics.
Our extensive experience allows us to guide you through the variety of available methods and define the optimal analytical strategy for your project.
Antibody CQAs and Analytical Techniques
Critical Quality Attribute |
Biofidus Techniques |
Development Purpose |
|---|---|---|
| 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.
