Characterization for the Similarity Assessment between Proposed Biosimilar SB12 and Eculizumab Reference Product Using a State-of-the-Art Analytical Method

2.1 Reference Products

The EU- and US-sourced eculizumab RP, which expired from January 2016 to October 2022, was purchased from local distributors and then stored according to the manufacturer’s instructions.

Up to 49 EU-sourced RPs and 25 US-sourced RPs, which expired from January 2016 to October 2022, were purchased from local distributors and analyzed (refer to similarity assessment plan). Multiple lots of EU- and US-sourced RPs were procured over time and stored, handled according to the manufacturer’s instructions.

2.2 Intact Mass

Intact mass analysis was performed to determine MW by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS).

Glycosylated and deglycosylated whole protein mass, glycosylated and deglycosylated HC mass, and LC mass of eculizumab (SB12 and RP) were measured using a mass spectrometer after treatment or non-treatment of 1 M 1,4-Dithiothreitol (DTT) and/or PNGase F (NEB). The digested samples were loaded on a BEH300 C4 reverse-phase column (Waters) connected to Waters ultra performance liquid chromatography (UPLC)-MS. Data acquisition and processing was performed using MassLynx v4.1 (Waters) and/or BiopharmaLynx v1.2 software (Waters).

2.3 Peptide Mapping

Peptide mapping, which involves enzymatic digestion and tandem MS, was performed to analyze the overall amino acid sequence, post-translational modifications (oxidation and deamidation, etc.), and disulfide bond by LC-ESI-MS.

Eculizumab (SB12 and RP) samples were solubilized with 8 M urea. The denatured samples were reduced by 1 M DTT, and 1 M iodoacetamide (IAA) was then added for alkylation. The samples were 50 mM Tris-HCl buffer (pH 7.8) exchanged for enzyme digestion and digested with the endoprotease trypsin (Roche). For the disulfide linkage analysis, the reducing step was not performed and the non-reduced samples were also digested with trypsin. The digested samples were loaded on a BEH300 C18 reverse-phase column (Waters) connected to Waters UPLC-MS. Data acquisition and processing was performed using MassLynx v4.1 (Waters) and/or BiopharmaLynx v1.2 software (Waters).

2.4 Size Exclusion Chromatography Coupled with Multi-Angle Light Scattering

Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) is used to determine the molar mass and extinction coefficient of the soluble proteins in eculizumab (SB12 and RP). Molar mass calculation was performed on a portion of the data channels near the peak apex. Extinction coefficient was determined by refractive index signal in MALS.

2.5 Circular Dichroism Spectroscopy

For far-ultraviolet (UV) and near-UV circular dichroism (CD) studies, eculizumab (SB12 and RP) samples were diluted with formulation buffer. A Chirascan Q100 (Applied Photophysics) with a 0.1 mm path length cell for far-UV and 10 mm path length cell for near-UV was used. The observed CD signal within a 200–260 range for a far-UV CD scan and a 250–350 range for a near-UV scan was blank-substracted and the average of the triplicate scans was used to make the CD plot. The CDNN algorithm was used to fit the far-UV CD data for prediction of the secondary structure.

2.6 Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet iS50 FT-IR spectrophotometer (Thermo Scientific) equipped with a Smart Orbit diamond attenuated total reflection (ATR) accessory and the MONIC software package for spectrophotometric control and data analysis.

2.7 Differential Scanning Calorimetry

Nano-differential scanning calorimetry (DSC; TA Instruments) was used to analyze the melting temperature and thermodynamics of eculizumab (SB12 and RP). For DSC analysis, eculizumab was diluted with formulation buffer. The diluted sample and corresponding formulation buffer were loaded into the analyzer. Thermodynamic scanning was performed from 15 to 100 °C and the scan rate was 1.5 °C/min. Data processing was performed by NanoAnalyze software (TA Instruments).

2.8 Sedimentation Velocity-Analytical Ultra Centrifugation

The sedimentation velocity-analytical ultra centrifugation (SV-AUC; Beckman) method is used to identify and characterize monomer content, the presence of aggregates and fragments, and the MW of the main molecular species in a protein solution. Eculizumab (SB12 and RP) samples were diluted to 0.4 mg/mL with formulation buffer. Centrifugation was carried out at 20 °C at a speed of 45,000 rpm. Radial scans of the concentration profile were collected sequentially by absorbance at 280 nm until no further sedimentation was observed. The resulting data sets were analyzed using the SEDFIT program, with a continuous c(s) distribution model, yielding best-fit distributions for the number of sedimenting species and the effective MWs.

2.9 Hydrogen/Deuterium Exchange-Mass Spectrometry

Hydrogen/deuterium exchange-MS (H/DX-MS) was initiated by dilution of eculizumab (SB12 and RP) in D2O buffer for 10 s, 1 min, 10 min, 1 h and 4 h. After labeling, the solution was mixed with quenching buffer and injected into the Waters nano LC system. Loaded protein was digested on an immobilized pepsin column (Waters) and the digested peptide fragments was separated on an analytical C18 column (Waters). The analyte was then introduced to MS with MSE mode. Mass spectra were analyzed using PLGS software (Waters) for peptide identification, and DynamX software (Waters) for deuterium uptake calculation and generation of the butterfly plot and uptake plots.

2.10 Size Exclusion-High Performance Liquid Chromatography

For SEC analysis, eculizumab (SB12 and RP) was injected onto a TSK-GEL G3000 SWXL analytical column (Tosoh) connected to a Waters HPLC system. The mobile phase (100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8) was used as the isocratic gradient. Monomer and high MW (HMW) were detected by UV signal at 280 nm. Data were acquired and processed by Empower®3 software (Waters).

2.11 Capillary Electrophoresis-Sodium Dodecyl Sulfate

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) was performed using the capillary electrophoresis system PA800 plus Pharmaceutical Analysis (SCIEX). Eculizumab (SB12 and RP) samples were mixed with 250 mM IAA, 10 kDa internal standard and the SDS-MW sample buffer (Beckman) under non-reducing or reducing conditions. For the reduced condition, IAA was replaced with 14.3 M 2-mercaptoethanol. The mixture was incubated in a heated condition for denaturation and loaded for separation in the capillary cartridge. Electrophoresis was performed and the signals were monitored at 220 nm using 32 Karat software (SCIEX).

2.12 Imaged Capillary Isoelectric Focusing

The imaged capillary isoelectric focusing (icIEF) system performs free solution isoelectric focusing in a capillary column and detects focused protein zones using a whole-column UV absorption detector. Eculizumab (SB12 and RP) samples were mixed with pharmalyte 3-10, methyl cellulose, urea, distilled water, and pI marker. The mixtures were loaded and analyzed onto an iCE3 instrument (Protein Simple).

2.13 Anion Exchange Chromatography

Eculizumab (SB12 and RP) was injected onto a weak anion exchange (WAX) analytical column (Thermo Scientific) connected to a Waters HPLC system. 2-(N-morpholino)ethanesulfonic acid (MES) buffer and MES containing sodium chloride buffer were used as the mobile phases A and B, respectively, for anion exchange chromatography. Charge variants were detected by UV signal at 280 nm. Data were acquired and processed using Empower®3 software (Waters).

2.14 Glycan Profile

The identification and relative percentage of N-linked glycan in eculizumab was determined by hydrophilic interaction UPLC (HILIC-UPLC). Eculizumab (SB12 and RP) samples were treated with PNGase-F enzyme to release N-glycan after denaturation. The released N-glycan was separated from proteins using cold ethanol, and dried completely. 2-aminobenzamide (2-AB)-labeled N-glycan was gradually separated using an Acquity UPLC BEH glycan column (Waters), and quantified on a fluorescence detector connected to a Waters UPLC system. For identification, N-glycan was labeled with procainamide and identified on LC-ESI-MS connected to a Waters UPLC system.

2.15 Reverse Phase-Ultra Performance Liquid Chromatography

The hydrophobicity profile of eculizumab (SB12 and RP) samples was determined using 2.15 reverse phase-UPLC (RP-UPLC). Eculizumab (SB12 and RP) samples were loaded on a BEH300 C4 reverse-phase column (Waters) and separated by reverse-phase mode on UPLC (Waters) with UV detection at 280 nm. Data were acquired and processed using Empower®3 software (Waters).

2.16 Protein Concentration by UV A280

Protein concentrations of eculizumab (SB12 and RP) were determined by UV measurement at A280. Eculizumab (SB12 and RP) samples were diluted with 0.9% sodium chloride to 1.0 mg/mL based on the expected concentration of each sample. Absorbance was measured at a wavelength of 280 nm after normalizing with 0.9% sodium chloride (blank solution).

2.17 C5 Inhibition Assay

Eculizumab (SB12 and RP) inhibits the formation of the membrane attack complex (MAC) by blocking C5 cleavage, and prevents B-cell lysis promoted by the anti-CD20 antibody. The blockage effect of eculizumab on the terminal complement complex is evaluated through the detection of cell lysis induced by complement dependent cytotoxicity (CDC) activation. To determine the effect of eculizumab (SB12 and RP), CDC activation is induced through human serum as a complement and anti-CD20 antibody, and the effect of blocking cell lysis is measured through incubation with Raji cells (ATCC), which are CD20-positive B cells.

2.18 Anti-Hemolytic Assay

Eculizumab (SB12 and RP) inhibits cleavage of C5 to C5a and C5b and prevents generation of the terminal complement complex C5b-9 (MAC) responsible for the lysis of paroxysmal nocturnal hemoglobinuria/atypical hemolytic uremic syndrome (PNH/aHUS) red blood cells (RBCs) lacking cell surface terminal complement inhibitor CD59. The anti-hemolytic assay using the luminescence system measured the inhibition of hemolysis of RBCs (Innovative Research) by eculizumab (SB12 and RP)

2.19 C5 Binding Assay

Human complement protein C5 is the first material adsorbed onto the solid phase. Blocking of non-specific binding to the plate surface is followed using BSA-containing buffer. After blocking, eculizumab (SB12 and RP) binds to C5, and a peroxidase-linked secondary antibody then binds to eculizumab (SB12 and RP). Finally, appropriate chromogenic substrate is added, producing colored product. The test plate is read on a spectrophotometer at 450 nm, yielding the optical density that is directly proportional to the concentration of eculizumab (SB12 and RP). Finally, relative C5 binding activity of eculizumab (SB12 and RP) is calculated by analyzing the four-parameter curve plotted from the data in the parallel-line analysis (PLA) software (Stegmann Systems GmbH).

2.20 FcRn Binding Assay by Surface Plasmon Resonance (SPR)

Purified FcRn is immobilized on a CM5 sensor chip (Cytiva) using N-Hydroxysuccinimide/1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide. Various concentrations (31.7–507 nM) of SB12 and eculizumab (SB12 and RP) samples were prepared by serial dilution with Hepes buffered saline–EDTA, Surfactant P20 buffer. Samples were injected into the flow cell for 120 s association at a flow rate of 30 μL/min, and 240 s dissociation at a flow rate of 30 µL/min. Binding affinity was calculated for the sensorgrams using BIAevaluationTM software’s 1:1 binding model.

2.21 Additional Biological Characterization

In order to measure Fc gamma receptor affinity for eculizumab (SB12 and RP; by surface plasmon resonance [SPR]), the His-tagged Fc gamma receptor is captured by the immobilized anti-His antibody on the sensor chip surface, and a series of diluted eculizumab in free solution is then injected onto the captured Fc gamma receptor. The association is followed by running buffer and dissociation. Afterwards, regeneration solution is injected, thus bound eculizumab and Fc gamma receptor are washed out for the sequential cycle. The association and dissociation of serially diluted eculizumab are evaluated to the appropriate binding model.

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