Table 1.

Attributes and analytical techniques for characterization of ABP 980 and trastuzumab reference product

Category	Analytical techniques and attributes
Primary structure	Whole mass by mass spectrometry: intact molecular mass and mass spectrum profile Reduced and deglycosylated molecular masses of HC and LC: molecular masses of HC and LC and mass spectrum profile Reduced peptide map: amino acid sequence, peptide map profile, post-translational modifications Non-reduced peptide map: disulfide structure and peptide map profile Glycan map by HILIC HPLC: afucosylation, high mannose, galactosylation, afucosylated galacosylation, sialylation, glycan map profile cIEF: isoelectric point, profile Amino acid analysis: extinction coefficient Identity by ELISA
Higher-order structure	FTIR: spectral similarity, spectral profile Near UV CD: spectral similarity, spectral profile DSC: Tm1, Tm2, profile
Particles and aggregates	HIAC: ≥ 2 µm, ≥ 5 µm, ≥ 10 µm, ≥ 25 µm particles MFI: ≥ 5 µm particles and ≥ 5 µm non-spherical particles SV-AUC: HMW, profile SE-HPLC–LS: Molar mass, profile
Product-related substances and impurities	SE-UHPLC: HMW, main peak, LMW, profile rCE-SDS: HC + LC, LMW + MMW, NGHC, profile nrCE-SDS: pre-peaks, main peak, profile
Thermal stability and degradation	SE-HPLC: HMW, main peak degradation rCE-SDS: HC + LC, LMW + MMW, NGHC degradation nrCE-SDS: pre-peak degradation Proliferation inhibition bioassay: potency degradation
General properties	Protein content: protein content, reconstituted protein concentration Reconstitution time
Process-related impurities	HCP ELISA 2D-DIGE LC/MS Protein A ELISA qPCR
Fab-mediated biological activity	Potency: proliferation inhibition (BT-474 cells) HER2 binding: ELISA, SPR Inhibition of AKT phosphorylation Proliferation inhibition (NCI-N87 cells) Proliferation inhibition synergy with chemotherapeutic (NCI-N87 cells) Lack of proliferation inhibition (non-amplified HER2 MCF7 cells)
Fc-mediated biological activity	FcRn binding FcγRIIIa (158 V) binding FcγRIIIa (158F) binding FcγRIa binding FcγRIIa (131H) binding FcγRIIb binding FcγR binding on primary macrophages FcγRIIIb binding C1q binding
Fab and Fc-mediated biological activity	NK92 ADCC activity PBMC ADCC activity Lack of ADCC (HER2-negative cells) ADCP activity Lack of CDC activity

2D-DIGE two-dimensional differential in-gel electrophoresis, ADCC antibody-dependent cellular cytotoxicity, ADCP antibody-dependent cellular phagocytosis, CDC complement-dependent cytotoxicity, cIEF capillary isoelectric focusing, DSC differential scanning calorimetry, ELISA enzyme-linked immunosorbent assay, FcγR Fc gamma receptor, FcγRIa Fc gamma receptor type 1a, FcγRIIa Fc gamma receptor type 2a, FcγRIIb Fc gamma receptor type 2b, FcγRIIIa Fc gamma receptor type 3a, FcRn Fc neonatal receptor, FTIR Fourier-transformed infrared, HC heavy chain, HER2 human epidermal growth factor receptor 2, HILIC hydrophilic interaction liquid chromatography, HMW high molecular weight, HPLC high-performance liquid chromatography, LC light chain, LC/MS liquid chromatography with mass spectrometry, LMW low molecular weight, MFI micro-flow imaging, NGHC non-glycosylated heavy chain, nrCE-SDS non-reduced capillary electrophoresis with sodium dodecyl sulfate, PBMC peripheral blood mononuclear cells, qPCR quantitative polymerase chain, reactionrCE-SDS reduced capillary electrophoresis with sodium dodecyl sulfate, SDS sodium dodecyl sulfate, SE-HPLC–LS size-exclusion high-pressure liquid chromatography–light scattering, SE-UHPLC size-exclusion ultra high-performance liquid chromatography, SPR surface plasmon resonance, SV-AUC sedimentation velocity analytical ultracentrifugation, UV CD ultraviolet circular dichroism