Antibody-Drug Conjugate (ADC) Analysis
Antibody-drug conjugates are among the most dynamic targeted cancer therapies, designed to deliver highly potent cytotoxic payloads specifically to tumor cells while limiting effects on healthy tissue. This requires a precise understanding of antibody structure, payload conjugation, target-binding properties as well as plasma stability.
At Biofidus, we offer end-to-end antibody-drug conjugate (ADC) analysis and characterization services covering both structural and functional characterization to build a robust, regulator-ready data package for your ADC project.
Our analytical portfolio includes in-depth protein primary structure analysis, post-translational modification profiling, determination of drug-to-antibody ratio (DAR) and DAR distribution, assessment of conjugation sites, level of aggregation and free payload, as well as stability and forced-degradation studies to evaluate linker and payload stability under relevant conditions.
Functionally, we characterize your lead ADC molecules using customized cell-based potency assays, target-binding and kinetics by surface plasmon resonance (SPR) or related technologies, and, where needed, assays that probe internalization and mechanism of action.
Our vast experience and deep knowledge in ADC analytics allow us to guide you through the variety of available methods and to define the optimal analytical strategy for your antibody-drug conjugate.
Antibody CQAs and Analytical Techniques
| Critical Quality Attribute | Biofidus Techniques | Development Purpose |
|---|---|---|
| Drug-to-antibody ratio & distribution | HIC, LC-MS, UV/Vis | Average DAR, DAR distribution, heterogeneity |
| Conjugation sites & site occupancy | Peptide mapping LC-MS/MS | Conjugation sites, occupancy, positional heterogeneity |
| ADC internalization | Cell-based internalization assays | Cellular uptake, trafficking, target-dependent internalization |
| 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 |
In accordance with your needs, we adapt and, if required, qualify our platform methods according to ICH guidelines, tailoring them to your drug substance (DS), its formulation matrix and your specific analytical questions.
Get in contact with us to quickly identify the optimal setup for your ADC analytical challenge and to speak directly from expert to expert.
