Table 3.
Product parameters and test methods applied to analysis of all batches of each IC.
| Product parameter | Test method | Purpose |
|---|---|---|
| Identity | Tryptic peptide map, HPLC, UV, and MS detection | Identification test to evaluate similar peptides and examine for new different species |
| Characterization of peptide masses, and assess intact N- and C-terminal amino acid residues, distribution of O-linked glycans on specific peptides | ||
| SDS-PAGE silver stain (reducing /non-reducing) | Identification test to evaluate principal bands for monomer, dimer, aggregates, and clips | |
| Strength | UV spectrophotometric scan | Strength test to measure protein concentration |
| Purity and impurities | HIC | Purity test to evaluate hydrophobicity and assess level of misfolded species |
| HCP ELISA | Impurities derived from CHO cells are quantified by ELISA | |
| Protein A ELISA | Impurities derived from leached Protein A column are quantified | |
| SE-HPLC | Purity test to quantify aggregated species | |
| SDS-PAGE (Coomassie) scanning densitometry | Purity test to quantify protein aggregate and clips | |
| Potency | Apoptosis bioassay | Cell-based bioassay based on potency to prevent induced apoptosis in cells (in vitro neutralization of TNF) |
| Quality | Sialic acid | Test to quantify total N- and O-linked sialic acid content |
| Receptor-binding assay | Test for strength of binding to target molecule, TNF | |
| N-linked oligosaccharide map | HPLC test for presence and relative % of specific N-linked glycansa | |
| Charge heterogeneity | IEF | Characterization test to evaluate charge heterogeneity |
| Purity | dSE-HPLC | Characterization test to quantify specific low-molecular-weight clipped species |
| Negative charge heterogeneity | AEX | Characterization test to evaluate negative charge heterogeneity |
Each etanercept monomer has three sites for the addition of N-linked oligosaccharide structures. Each of the three N-linked oligosaccharides added to etanercept can be any one of multiple characterized species.
AEX, anion-exchange chromatography; dSE-HPLC, denaturing size exclusion HPLC; ELISA, enzyme-linked immunosorbent assay; HCP, host-cell protein; HIC, hydrophobic interaction chromatography; HPLC, high-performance liquid chromatography; IC, intended copy; IEF, isoelectric focusing; MS, mass spectrometry; PRI, process-related impurities; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE-HPLC, size exclusion HPLC; TNF, tumor necrosis factor; UV, ultraviolet.