Table I.
Similarity Testing Plan and the Analytical Methods for the Structural and Functional Characterization of the Proposed Biosimilar ABP 710 and Infliximab Reference Products
| Category | Attributes and Analytical Techniques |
|---|---|
| General properties | Protein content by gravimetric analysis |
| Reconstituted protein concentration by UV absorbance | |
| Reconstitution time | |
| Primary structure and post-translational modifications | Intact molecular mass analysis |
| Reduced and deglycosylated molecular masses of HC and LC | |
| Protein sequence by reduced peptide map | |
| Reduced peptide map, post-translational modifications: levels of deamidation and oxidation | |
| Disulfide structure by nonreduced peptide map | |
| Glycan map by HILIC | |
| Isoelectric point by capillary isoelectric focusing | |
| Identity by anti-idiotype ELISA | |
| Higher order structure | Secondary structure by FTIR spectroscopy |
| Tertiary structure by NUV-CD spectroscopy | |
| Conformation and thermal stability by DSC | |
| Product-related substances and impurities | Size variants by SE-UHPLC, rCE-SDS, and nrCE-SDS |
| Charge variants by CEX-HPLC and CEX-HPLC after carboxypeptidase B treatment | |
| Particles and aggregates | Subvisible particles by HIAC and MFI |
| Submicron particles by FFF and DLS | |
| Solution state size distribution by SV-AUC | |
| Molar mass of size variants by SE-HPLC-LS | |
| Biological activity | Inhibition of sTNFα-induced apoptosis in U937 |
| Binding to sTNFα by ELISA | |
| Binding to sTNFα by SPR | |
| Binding kinetics to sTNFα by SPR | |
| Inhibition of sTNFα-induced IL-8 release in HUVEC | |
| Inhibition of sTNFα-induced cell death in L929 | |
| Binding to mbTNFα on CHO MT-3 cells by imaging cytometry | |
| Reverse signaling in Jurkat mbTNFα line | |
| Binding to LTα by SPR | |
| Inhibition of LTα-induced IL-8 release in HUVEC | |
| Binding to FcγRIIIa (158V) by SPR | |
| Binding kinetics to FcγRIIIa (158V) by SPR | |
| Binding to FcγRIIIa (158F) by SPR | |
| Binding to primary NK cells by FACS | |
| NK92 ADCC activity | |
| PBMC ADCC activity | |
| Binding to C1q by ELISA | |
| CDC activity | |
| Binding to FcγRIIa (131R) by SPR | |
| ADCP activity | |
| Binding to FcγRIa by AlphaLISA | |
| Binding to FcγRIIb by SPR | |
| Binding to FcγRIIIb by SPR | |
| Binding to FcRn by AlphaScreen | |
| Binding to FcRn by SPR | |
| Thermal stability and forced degradation | Reconstituted forced degradation study at 40°C |
| Lyophilized stressed degradation study at 40°C | |
| Lyophilized accelerated degradation study at 25°C assessedby purity and potency assays |
ADCC antibody-dependent cell-mediated cytotoxicity, ADCP antibody-dependent cellular phagocytosis, SV-AUC sedimentation velocity analytical ultracentrifugation, CDC complement-dependent cytotoxicity, CEX-HPLC cation exchange high performance liquid chromatography, CHO Chinese Hamster Ovary cell, cIEF,capillary isoelectric focusing, C1q the first subcomponent of the C1 complex of the classical pathway of complement activation, DLS dynamic light scattering, DSCdifferential scanning calorimetry, ELISA enzyme-linked immunosorbent assay, FACS fluorescence-activated cell sorting, FcR fragment crystallizable receptor, FcγRIaFc gamma receptor Type Ia, FcγRIIa Fc gamma receptor Type IIa, FcγRIIb Fc gamma receptor Type IIb, FcγRIIIa Fc gamma receptor Type IIIa, FcγRIIIb Fc gammareceptor Type IIIb, FcRn neonatal Fc receptor, FFF field flow fractionation, FTIR Fourier-transform infrared spectroscopy, HC heavy chain, HCP host cell protein,HIAC high accuracy light obscuration, HILIC hydrophilic interaction liquid chromatography, UHPLC untra high performance liquid chromatography HPLC highperformance liquid chromatography, HUVEC human umbilical vein cells, LC light chain, mbTNF membrane bound tumor necrosis factor, MFI micro-flow imaging,NUV-CD near-ultraviolet circular dichroism, nrCE-SDS non-reduced capillary electrophoresis–sodium dodecyl sulfate, PBMC peripheral blood mononuclear cell,rCE-SDS reduced capillary electrophoresis–sodium dodecyl sulfate, SE-HPLC-SLS size exclusion high performance liquid chromatography with light scattering, SE-HPLC size exclusion high performance liquid chromatography, SPR surface plasmon resonance, sTNF soluble tumor necrosis factor