Table 3.
An overview of the characterization of mAb biosimilars (Visser et al., 2013)
| Category | Quality attribute | Methods |
|---|---|---|
| Physicochemical characterization | ||
| Primary structure | Amino acid sequence | Red. RP‐HPLC–ESI–MS peptide mapping, intact mass of whole mAb, HC and LC by RP‐HPLC–ESI–MS, Red. RP‐HPLC‐UV peptide mapping |
| Higher order structure | Disulfide bridging Free thiols Secondary and quaternary structure Thermodynamic stability | Non‐red. RP‐HPLC‐ESI–MS peptide mapping Ellman's assay CD, FTIR, HDX‐MS, X‐ray DSC |
| General charge heterogeneity and amino acid modifications | 0 K variant, acidic variants, basic variants, Gln‐variant, Lys‐variant, amidated proline Glycation Oxidation/deamidation/C‐terminal variants | CEX digested/undigested Boronate affinity RP‐HPLC‐UV/MS peptide mapping |
| Glycosylation | Galactosylation, sialylation, mannosylation, afucosylation, bisecting GlcNAc, NGNA, α‐galactose, qualitative glycosylation pattern | NP‐HPLC‐FL |
| Size heterogeneity | Monomer, low molecular weight (LMW) and high molecular weight (HMW) variants (aggregates) Heavy chain (HC), light chain (LC), aglycosylated HC, clipped variants Monomer, LMW (e.g., half antibodies and HHL variant) and HMW variants Subvisible particles Visible particles | SEC, AF4 Red. CE‐SDS Non‐red. CE‐SDS Light obscuration (PhEur, ≥10 μm and >25 μm) Visual inspection (PhEur) |
| Functional characterization | ||
| Target and receptor binding | FCRn binding SPR FcγR binding (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(F158), FcγRIIIa(V158), FcγRIIIb) SPR | |
| Bioactivity | CD20 target binding CDC potency ADCC potency Apoptosis | Cell‐based binding assay Cell‐based CDC assay Cell‐based ADCC assay Cell‐based apoptosis assay |
ADCC, antibody‐dependent cell‐mediated cytotoxicity; AF4, asymmetric flow‐field fractionation; CD, circular dichroism; CDC, complement dependent cytotoxicity; CE‐SDS, capillary electrophoresis with sodium dodecyl sulfate; CEX, cation‐exchange chromatography; DSC, differential scanning calorimetry; ESI, electrospray ionization mass; FL, fluorescence; FTIR, fourier transform infrared; HDX, hydrogen deuterium exchange; HPLC, high‐performance liquid chromatography; LC, liquid chromatography; mAb, monoclonal antibody; MS, mass spectrometry; NP, normal phase; RP, reverse phase; SEC, size‐exclusion chromatography; SPR, surface plasmon resonance; UV, ultraviolet.
Reproduced from Visser et al. (2013).