Table 1.
Analytical methods used for evaluating similarity of HLX02 biosimilar to its innovator Herceptin®
| Test classification | Quality attributes | Tier | Methods | Demonstrated similarity |
|---|---|---|---|---|
| Physicochemical properties and purity | ||||
| Primary structure | Amino acid sequence | I | Reduced peptide mapping by LC–MS/MS | Identical to the RP |
| III | Amino acid analysis by RP-UPLC | Similar to the RP | ||
| Molecular weight of intact, reduced antibody and subunits | II | Intact protein mass analysis by LC–MS | Same mass species | |
| II | Reduced protein mass analysis by LC–MS | |||
| Reduced and deglycosylated mass analysis by LC–MS | ||||
| II | Papain digested protein subunit mass analysis by LC–MS | |||
| Disulfide linkage | II | Non-reduced peptide mapping by LC–MS/MS | Identical to the RP | |
| Free thiols | II | Free thiol fluorescent detection kit | Similar to the RP | |
| PTMs | II | Reduced peptide mapping by LC–MS/MS | Similar to the RP | |
| Glycosylation site | II | Deglycosylated and reduced peptide mapping | Identical to the RP | |
| Higher order structure | Secondary and tertiary structure | III | DSC | Similar to the RP |
| CD | ||||
| FLR | ||||
| FTIR | ||||
| NMR | ||||
| Charge variants | Charge variants | II | CEX | Similar to the RP; slightly less peak 4 |
| Isoelectric point | II | icIEF | Identical to the RP | |
| Size variants | Aggregates, monomers, LMWs and NGHC | II/III | SEC-HPLC | Similar to the RP; slightly less aggregates and NGHC |
| Reduced and non-reduced CE-SDS | ||||
| SEC-MALS | ||||
| SV-AUC | ||||
| Glycosylation | Glycan distribution | II | HILIC UPLC-FLD | Similar to the RP; slightly higher sialylation, not clinically meaningful |
| Sialic acid | II | RP HPLC-FLD | ||
| Monosaccharide analysis | III | Ion chromatography | ||
| Functional assays | ||||
| Immunochemical properties | FcRn, FcγR (FcγRIa, FcγRIIa, FcγRIIb/c, FcγRIIIa (V), FcγRIIIa (F), FcγRIIIb) | II | SPR | Similar to the RP |
| FcγRIIIa | II | FcγRIIIa affinity chromatography | Similar to the RP | |
| C1q | III | ELISA | Similar to the RP | |
| Bioactivity | HER2 binding | I | ELISA | Similar to the RP |
| SPR | Similar to the RP | |||
| Anti-proliferation | I | Cell-based assay | Similar to the RP | |
| Apoptosis | II | Cell-based assay | Similar to the RP | |
| ADCC | I | Cell-based assay | Similar to the RP | |
| CDC | III | Cell-based assay | Similar to the RP, both negative | |
| Process-related impurities | DNA | I | qPCR | Similar to the RP |
| HCP | I | ELISA | Similar to the RP | |
| Protein A | I | ELISA | Similar to the RP | |
| Particles | Sub-micro particles | III | DLS | Similar to the RP |
| Sub-visible particles | III | MFI | Similar to the RP | |
ADCC antibody-dependent cell-mediated cytotoxicity, CD circular dichroism, CDC complement-dependent cytotoxicity, CE-SDS capillary electrophoresis–sodium dodecyl sulfate, CEX cation exchange chromatography, DLS dynamic light scattering, DSC differential scanning calorimetry, ELISA enzyme-linked immunosorbent assay, FcγR Fc gamma receptor, FcRn neonatal Fc receptor, FLR fluorescence spectrum, FTIR Fourier transform infrared spectroscopy, HCP host cell protein, HER2 human epidermal growth factor receptor 2, HILIC UPLC-FLD hydrophilic interaction–ultra performance liquid chromatography–fluorescence detection, icIEF imaged capillary isoelectric focusing, LC–MS/MS liquid chromatography tandem mass spectrometry, LMWs low molecular weight variants, MFI micro-flow imaging, NGHC non-glycosylated heavy chain, NMR nuclear magnetic resonance, PTMs post-translational modifications, qPCR quantitative real-time polymerase chain reaction, RP reference product, RP HPLC-FLD reversed-phase high-performance liquid chromatography–fluorescence detection, RP-UPLC reversed-phase ultra performance liquid chromatography, SEC-HPLC size-exclusion chromatography–high-performance liquid chromatography, SEC-MALS size-exclusion chromatography with multi-angle light scattering, SPR surface plasmon resonance, SV-AUC sedimentation velocity analytical ultracentrifugation