Physicochemical and Biological Characterization of the Proposed Biosimilar Tocilizumab

Table 1

Characterization strategy for the proposed biosimilar HS628 and originator tocilizumab.

Category	Attributes	Methods
Primary structure	Amino acid sequence	Reduced RP-UPLC-MS peptide mapping
	Molecular weight	RP-UPLC-MS
Higher order structure	Disulfide bridging	Nonreduced RP-UPLC-MS peptide mapping
	Free thiols	Ellman’s assay
	Secondary and tertiary structure	Circular dichroism (CD)
	Thermodynamic stability	Differential scanning calorimetry (DSC)
Posttranslational modifications	Deamidation, oxidation	Reduced RP-UPLC-MS peptide mapping
Charge heterogeneity	Charge variants	CEX-HPLC
	Isoelectric point	Imaged capillary isoelectric focusing (icIEF)
Size heterogeneity	High molecular weight impurities	SEC-HPLC
	Low molecular weight impurities	Nonreduced CE-SDS
	Nonglycosylated heavy chain (NGHC)	Reduced CE-SDS
Glycosylation	Glycan profile	NP-HPLC-FL
Binding activity	Affinity to IL-6R, FcγRIIIa, and FcRn	Surface plasmon resonance
Potency	Antiproliferative potency	Antiproliferation assay
	Signal transduction inhibition	Inhibition of STAT3 phosphorylation