Table 1.
Summarized quality attributes and key findings in physiochemical and biological similarity assessment between HLX01 and the reference products.
| Category | Product Quality Attributes | Tier | Analytical Methods | Assessment |
|---|---|---|---|---|
| Physiochemical characteristics | ||||
| Primary structure | Amino acid sequence | I | LC-MS/MS | Identical to the RP |
| Intact and reduced molecular weights | II | LC-MS | Similar to the RP | |
| Disulfide linkage pattern | II | LC-MS/MS | Identical to the RP | |
| Free thiol groups | II | Ellman’s assay | Similar to the RP | |
| PTMs：Deamidation, Oxidation, Glycosylation, C-terminal and N-terminal Variants | II | LC-MS/MS | Same modification sites and similar level of modifications to the RP | |
| Higher order structure | Protein secondary and tertiary structure | II/III | DSC, CD, FTIR | Similar to the RP |
| Charge heterogeneity | Charge heterogeneity | II | CEX icIEF |
Similar to the RP, with higher basic variants due to unprocessed HC C-terminal lysine, not clinical meaningful |
| Isoelectric point | II | icIEF | Same to the RP | |
| Glycosylation | N-linked glycans | II | RapiFluor labeling and HILIC UPLC-FLD | Same glycan types with slightly difference in %G0F + %Man + %Gal + % Afuc, not clinical meaningful |
| Sialylation | II | DMB labeling and RPLC-FLD | Similar to the RP | |
| N-linked glycosylation site, and site occupancy | II | LC-MS/MS | Same to the RP | |
| Molecular variants | Soluble aggregates | I | SEC-HPLC | Slightly lower than the RP |
| Monomer | II | SEC-HPLC, CE-SDS | Similar to the RP with high level of purity | |
| Low molecular weight fragment | II | SEC-HPLC, CE-SDS | Slightly lower than the RP | |
| Biological characteristics | ||||
| Immunological functions | FcRn binding activity | I | SPR | Similar to the RP |
| FcγRIa binding activity | II | SPR | Similar to the RP | |
| FcγRIIa binding activity | ||||
| FcγRIIb/c binding activity | ||||
| FcγRIIIa (F) binding activity | ||||
| FcγRIIIa (V) binding activity | ||||
| FcγRIIIb binding activity | ||||
| C1q binding activity | II | ELISA | Similar to the RP | |
| Antigen binding activity | I | FACS | Similar to the RP | |
| Biological activities | CDC activity | I | Cell based assay | Similar to the RP |
| ADCC activity | II | Cell based assay | Similar to the RP | |
| Apoptosis | II | Cell based assay | Similar to the RP | |
| Safety of residues | Residual DNA | I | PCR | Similar to the RP |
| Residual HCP | I | ELISA | Similar to the RP | |
| Residual Protein A | I | ELISA | Similar to the RP | |