FormalPara Key Summary Points

SB15 has been developed as a proposed biosimilar product of reference aflibercept (Eylea®), an approved biological drug product for retinal diseases including neovascular age-related macular degeneration (nAMD).

A comprehensive analytical characterization was conducted to assess the analytical similarity between SB15 and reference aflibercept (Eylea®) in terms of structural, physicochemical, and biological properties.

Based on the results, it was demonstrated that SB15 is highly similar to its reference products sourced from the United States (US-aflibercept) and European Union (EU-aflibercept), supporting safe and effective use of SB15.

Introduction

SB15 has been developed as a biosimilar to reference aflibercept (Eylea®, Regeneron Pharmaceutical Inc.), a recombinant fusion protein constructed by domain 2 from vascular endothelial growth factor receptor 1 (VEGFR1), domain 3 from VEGFR2, and the hinge region of the fragment crystallizable (Fc) domain of human immunoglobulin [1,2,3,4]. Aflibercept acts as a soluble decoy receptor with a higher binding affinity to VEGF isoforms such as VEFG-A, VEGF-B, and placental growth factors (PIGFs) compared to their natural receptors [5]. It mediates inhibition of the binding and activation of these cognate VEGF receptors [6,7,8,9]. Aflibercept has been approved by the United States (US) Food and Drug Administration (FDA), the European Medicines Agency (EMA), and other countries for a variety of ocular diseases, including neovascular age-related macular degeneration (nAMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), and diabetic retinopathy (DR) [3, 4, 10].

A biosimilar is a biologic product that is highly similar to its approved reference product (RP) without clinically meaningful differences in terms of the safety, purity, and potency [11,12,13]. Since all biologics, including biosimilars, are produced and extracted from living organisms and follow complex manufacturing processes, minor structural and functional differences between biosimilar products and corresponding RPs are inevitable [14]. For regulatory authorization, it must be investigated and demonstrated that these differences do not influence key quality attributes of the biosimilar and impacting its clinical safety and efficacy. In this aspect, guidelines of the US FDA and the EMA recommend a stepwise approach for biosimilar development, beginning with the demonstration of analytical similarity by comprehensive structural and functional characterization, followed by non-clinical and clinical studies [11, 15].

In the phase 3 clinical trial (SB15-3001; ClinicalTrials.gov: NCT04450329), equivalent efficacy and comparable safety, pharmacokinetics, and immunogenicity in participants with nAMD were demonstrated between SB15 and aflibercept RP in participants with nAMD [16, 17]. In this study, we focus on the comprehensive assessment of the analytical similarities between SB15 and its commercially available RP using product sourced from US and EU (US/EU-aflibercept). Multiple state-of-art analytical methods were used to enable detection and identification of differences in terms of the structural, physicochemical, and biological properties. Based on the risk assessment, quality attributes were evaluated by a range approach using pre-defined quality range with US/EU-aflibercept or side-by-side comparison. The results of the analytical similarity assessment demonstrate that the SB15 and US/EU-aflibercept are highly similar.

Methods

This article does not contain any studies with human participants or animals performed by any of the authors. All samples for the study were handled aseptically in a biological safety cabinet.

Reference Products

The commercially available aflibercept RP sourced from US and EU (US/EU-aflibercept) were purchased through local distributors. The purchased RPs were stored and handled according to the manufacturer’s instructions.

Mass Spectrometry-Based Analysis

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS) method was used for the analysis of primary structure and post-translation modification (PTM) analysis including N-glycan identification. An ACQUITY ultra performance liquid chromatography (UPLC, Waters, Milford, MA, USA) coupled to a Synapt G2 quadrupole time-of-flight mass spectrometer (Q-ToF, Waters, Milford, MA, USA) were used. The acquired data were processed using MassLynx v4.1 (Waters, Milford, MA, USA) and/or BiopharmaLynx v1.2 software (Waters, Milford, MA, USA).

Molecular Weight

Molecular weight was measured using LC–ESI–MS method in protein level. Since the aflibercept has high heterogeneity in N-glycan profile, SB15 and RP samples were deglycosylated and reduced by treating Dithiothreitol (DTT) and PNGase-F prior to LC–ESI–MS analysis. The BEH300 C4 column (Waters, Milford, MA, USA) was employed for the protein separation.

Peptide Identification and PTM Level Analysis

In the peptide level LC–ESI–MS analysis, samples were denatured and reduced by 8 M urea and 1 M DTT. After reduction, samples were alkylated using 1 M isodoacetamide (IAA) and buffer exchanged into the digestion buffer (1 M urea in 50 mM Tris–HCl, pH 7.5). Multiple enzymes including trypsin, Lys-C, and Asp-N were used for digestion. The BEH300 C18 column (Waters, Milford, MA, USA) was used for the peptide separation, while the disulfide linkage analysis reduction steps were ignored.

The chromatograms of SB15 and aflibercept RP were compared for the peptide mapping analysis. The amino acid sequence analysis, including N-terminal and C-terminal sequences, was performed using MS/MS-based peptide identification. Moreover, semi-quantitative analysis of PTM levels was conducted using the acquired data.

N-Glycosylation

N-glycan identification was performed by LC–ESI–MS method. To release N-glycan, samples were treated with PNGage-F after denaturation. Released N-glycans were separated from protein using ethanol precipitation and dried using EZ-2.3 vacuum evaporator (Genevac, Ipswich, EN, USA). Dried N-glycan samples were reconstituted with distilled water (DW), labeled by procainamide, and loaded onto a BEH amide column (Waters, Milford, MA, USA). N-glycan annotation was conducted by using GlycoWorkBench software.

For the N-glycan quantitation, 2-aminobenzamide (2-AB) was used for labeling of N-glycan. 2-AB labeled samples were separated through a BEH amide column (Waters, Milford, MA, USA), which is connected to UPLC system with fluorescence detector (Waters, Milford, MA, USA). Detection was done by fluorescence at λex = 330 nm and λem = 420 nm. Data were acquired and processed using Empower 3 software (Waters, Milford, MA, USA).

Circular Dichroism Spectroscopy

SB15 and RP samples were diluted with each formulation buffer (SB15: 7.78 mM sodium phosphate, 8% sucrose (w/v), 0.03% polysorbate 20 (w/v), pH 6.2; aflibercept RP: 10 mM sodium phosphate, 40 mM NaCl, 5% sucrose (w/v), 0.03% polysorbate 20 (w/v), pH 6.2) for the far-ultraviolet (UV) and the near-UV circular dichroism (CD) analysis. A Chirascan Q100 (Applied Photophysics, Leatherhead, UK) with a 0.1 mm path length cell (far-UV) and 10 mm path length cell (near-UV) was used. The far-UV CD scan and the near-UV CD scan were acquired within 200–260 nm and 250–350 nm range, respectively. The acquired CD spectra were blank-subtracted to the corresponding formulation buffer.

Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectroscopy of liquid formulations was performed on a Tensor 27 FT-IR spectrometer (Bruker Optics, Coventry, UK) by using the BioATR II attenuated total reflectance (ATR) unit. All samples were analyzed without dilution. The spectra were recorded at a controlled temperature of 25 °C from wave numbers of 4000–850 cm−1 with a resolution of 4 cm−1. The SB15 and RP samples were analyzed against the corresponding formulation buffer as background. Each measurement was an average of 60 scans. Sample spectra were recorded by using atmospheric compensation (elimination of disturbing H2O and/or CO2 bands in the result spectra). The second derivative of sample spectra was calculated with nine smoothing points and normalized by vector normalization. Data evaluation of secondary structure was performed with OPUS 7.5 QUANT2 software (Bruker Optics, Ettlingen, Germany) by using the alpha-helix and beta-sheet databases.

Differential Scanning Calorimetry

A MicroCal Auto VP-Capillary differential scanning calorimetry (DSC) system (Malvern Instruments. Malvern, UK) was used to analyze the melting temperature (Tm) of the SB15 and aflibercept RP samples. The samples and the corresponding buffer were heated from 20 to 100 °C with a heating rate of 60 °C/h. The μDSC cell was pressurized to prevent boiling of the sample during heating. All samples were diluted to 1 mg/ml in SB15 formulation buffer.

A baseline run was performed by loading the formulation buffer. Baseline was subtracted from each sample measurement. Thermal data was normalized for the protein concentration. The Tm values were determined at the center of the peak or shoulder by using derivative analysis of the heating scan. Data analysis was performed by using Origin DSC software (OriginLab, Northampton, MA, USA).

Hydrogen/Deuterium Exchange-Mass Spectrometry

SB15 and RP samples were diluted with D2O then incubated over 10 s, 1 min, 10 min, 1 h, and 4 h. After incubation, samples were quenched and injected onto a hydrogen/deuterium exchange-MS system (H/DX-MS) consisting of nanoACQUITY UPLC system including H/DX manager (Waters, Milford, MA, USA) and Synapt G2-Si Q-ToF MS (Waters, Milford, MA, USA) for online digestion and peptide level LC–MS analysis. A immobilized BEH pepsin column (Waters, Milford, MA, USA), BEH C18 VanGuard Pre-column (Waters, Milford, MA, USA), and BEH C18 analytical column (Waters, Milford, MA, USA) were installed on the H/DX-MS system. The analysis of data was performed by using PLGS software (Waters, Milford, MA, USA) for peptide identification, and DynamX software (Waters, Milford, MA, USA).

Size-Exclusion High-Performance Liquid Chromatography

SB15 and RP samples were injected onto a TSKgel G3000 SWXL column (Tosoh, Tokyo, Japan) attached to a 3.10 Size Exclusion-High-Performance Liquid Chromatography (SE-HPLC) system consisted of a Waters Alliance 2695 Separation Module (Waters, Milford, MA, USA) with a Waters 2487 Dual λ Absorbance Detector (Waters, Milford, MA, USA). The separated monomer and size impurities were detected at 280 nm. Data acquisition and processing were performed by Empower 3 software (Waters, Milford, MA, USA).

Capillary Electrophoresis-Sodium Dodecyl Sulfate

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis was performed under non-reduced and reduced condition. In non-reduced CE-SDS analysis, SB15 and aflibercept RP samples were mixed with 250 mM IAA, 10-kDa internal standard, and SDS-MW sample buffer and heated for 5 min at 70 °C. For reduced CE-SDS, 2-mercaptoethanol (BME) was used instead of IAA. After heating, samples were loaded onto a bare-fused silica capillary (Beckman Coulter, Fullerton, CA, USA) connected on PA 800 Plus CE system (Beckman Coulter, Fullerton, CA, USA). Electropherograms were obtained at 220 nm using 32 Karat software (SCIEX, Brea, CA, USA).

Imaged Capillary Isoelectric Focusing

Before the imaged capillary isoelectric focusing (icIEF) analysis, sialic acid of SB15 and aflibercept RP samples were removed by treating of sialidase A. After desialylation, samples were mixed with pharmalyte 3–10, pharmalyte 8–10.5, pI 6.61 marker, and pI 9.50 marker. The mixture was injected onto an icIEF cartridge (Protein Simple, San Jose, CA, USA) installed on iCE3 instrument (Protein Simple, San Jose, CA, USA). Data were obtained and processed using CFR (Protein Simple, San Jose, CA, USA) and Chrom perfect software (Protein Simple, San Jose, CA, USA), respectively.

VEGF-A 165/121/189 Binding Assay (ELISA)

Enzyme-linked Immunosorbent Assay (ELISA) is employed to determine VEGF-A 165/121/189 binding activity of aflibercept. VEGF-A 165 (293-VE/CF, R&D Systems, Minneapolis, MN, USA), VEGF-A 121 (4644-VS/CF, R&D System, Minneapolis, MN, USA) or VEGF-A 189 (8147-VE/CF, R&D System, Minneapolis, MN, USA) was absorbed onto a 96-well plate and then, BSA-containing buffer was treated into the plate for blocking non-specific binding. Aflibercept dilution series were added for binding to coated VEGF-A 165, 121 or 189 and then, the sequential addition of HRP-conjugated anti-hIgG (Fc specific) antibody, tetramethylbenzidine (TMB, T0440, Sigma-Aldrich, St. Louis, MO, USA) substrate, and 1 N sulfuric acid is followed. A measure to absorbance at a 450-nm wavelength can determine a relative binding activity of aflibercept to VEGF-A 165, 121 or 189. The VEGF-A 165/121/189 binding activities of aflibercept were calculated relative to a reference standard via a parallel line analysis (PLA) software (Stegmann Systems, GmbH, Rodgau, Germany).

HUVEC Anti-Proliferation (VEGF-A 165)

Human umbilical vein endothelial cells (HUVEC, C2519A, Lonza, Basel, Switzerland) was employed for the determination of a relative anti-proliferation potency of aflibercept. VEGF-A 165 (293-VE/CF, R&D Systems, Minneapolis, MN, USA) and aflibercept dilution were treated onto a microplate, and then, HUVEC in the endothelial cell medium (ECM, 1001, ScienCell) were sequentially treated. The proliferation of HUVEC was induced for 4 days at 37 °C, in 5% CO2 incubator. The changes of HUVEC growth were measured through Cell Titer-Blue® cell viability system (G8082, Promega, Madison, WI, USA) by quantifying the intensity of fluorescence dye activated by the metabolic process of live cells. The relative potency of HUVEC anti-proliferation by aflibercept was determined relative to a reference standard via a PLA software.

VEGF-A 165 Neutralization

VEGFR-2-overexpressed engineered cell line (NFAT-RE-Luc2P/KDR HEK293 cells, E8510, Promega) in the assay media (DMEM, 11995, Gibco, Grand Island, NY, USA) was added onto a microplate and, VEGF-A 165 (293-VE/CF, R&D Systems) was treated in turn. Aflibercept dilution series were added onto a plate and incubated for about 6 h at 37 °C, in 5% CO2 incubator to induce luciferase gene expression by VEGF signaling. A quantitation of a luminescence was measured in a dose-dependent manner through using a microplate reader (EnVision, E2104, Perkin Elmer, Waltham, MA, USA). A relative VEGF-A 165 neutralization potency was calculated and determined relative to a reference standard via a PLA software.

FcRn/PlGF-1/PlGF-2/VEGF-B 167 Binding (SPR)

Surface Plasmon Resonance (SPR) is employed to evaluate neonatal fragment crystallizable receptor (FcRn) binding affinity of aflibercept. Recombinant human FcRn (8639-FC, R&D Systems, Minneapolis, MN, USA), PlGF-1 (264-PGB, R&D Systems, Minneapolis, MN, USA), PlGF-2 (6837-PL, R&D Systems, Minneapolis, MN, USA), or VEGF-B 167 (751-VE/CF, R&D Systems, Minneapolis, MN, USA) was immobilized on CM5 chip (BR-1005-30, Cytiva). Aflibercept dilution series was prepared using HBS-EP buffer. Diluted aflibercept was injected into a flow cell and dissociation was conducted. The kinetic constants were calculated by the sensorgrams regressed to 1:1 binding model of BIAevaluation™ software.

Results

The analytical similarity between SB15 and the US/EU-aflibercept was evaluated by the comprehensive analytical characterization using state-of-the-art methods. Analyses were implemented for the primary structure, higher-order structure, physicochemical properties, and functional activities. The list of analytical methods employed in the similarity assessment is provided in Table 1.

Table 1 List of quality attributes and analytical methods for the analytical characterization
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Primary Structure

Aflibercept is a recombinant fusion glycoprotein composed of the second domain of VEGFR-1, the third domain of the human VEGFR-2, and the Fc domain of human immunoglobulin G (IgG). Aflibercept consists of polypeptide chain linked with disulfide bonds and has five N-glycosylation sites on each chain. Due to its heterogeneity of N-glycan species on five N-glycosylation sites, it is difficult to accurately measure the molecular weight of the intact protein. Therefore, the molecular weights of SB15 and US/EU-aflibercept were compared for the deglycosylated and reduced samples. All experimental values for both SB15 and US/EU-aflibercept were equal to the theoretical values within 0.01%. In addition, identified disulfide linked peptide of SB15 were identical to aflibercept RP (Table 2). Using site-specific N-glycosylation analysis, it was also confirmed that SB15 and aflibercept RP have similar predominant N-glycan for all five N-glycosylation sites (data not shown). Based on the results of single chain molecular weight, disulfide linkage, and site-specific N-glycosylation analysis, the intact molecular weight of SB15 and US/EU-aflibercept were considered comparable.

Table 2 Identified disulfide linked peptides and their m/z for representative SB15 and US/EU-aflibercept
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The peptide map by trypsin, Lys-C, and Asp-N were compared and amino acid sequence were confirmed using MS/MS data. As shown in Fig. 1, SB15 and US/EU-aflibercept showed similar profile without any missing or newly observed peaks. The amino acid sequences were confirmed as identical with 100% sequence coverage (data not shown). For all three products, the pre-dominant C-terminal variant was the lysine-removed form and only one N-terminal sequence was identified. The methionine oxidation level and asparagine deamidation level of SB15 showed slight differences compared to US/EU-aflibercept (data not shown). However, no meaningful differences were observed in biological activities (Table 3). Even in the oxidative and basic stressed samples, biological activities were not changed regardless of increased oxidation and deamidation levels, respectively (data not shown).

Fig. 1
figure 1

A–C Overlay of peptide mapping profiles of SB15 (red), US-aflibercept (green), and EU-aflibercept (blue) after trypsin (A), Lys-C (B), and Asp-N (C) treatment

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Table 3 Summarized results of biological quality attributes
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Higher-order Structures

The higher-order structures of SB15 and US/EU-aflibercept were compared using various orthogonal methods. In the far-UV CD, minimum and maximum wavelength of spectrum and their residue molar ellipticity of SB15 were comparable to US/EU-aflibercept (Fig. 2A). Also, the resulting relative contents of secondary structural features were comparable (data not shown). The near-UV CD spectrum showed no meaningful difference in terms of profile and maximum wavelength between the three products which support the similarity in tertiary structure (Fig. 2B).

Fig. 2
figure 2

A–D Comparison for higher-order structure profile of SB15 and US/EU-aflibercept. A Far-UV CD spectra, B near-UV CD spectra, C second derivative FT-IR spectra, D DSC thermograms. CD circular dichroism, DSC differential scanning calorimetry, FT-IR Fourier transform-infrared spectroscopy, UV ultraviolet

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The secondary structure was additionally characterized by using FT-IR spectroscopy. The second derivative FT-IR spectra of SB15, and US/EU-aflibercept, showed similar profiles which overlap almost of amide I and amide II region, suggesting the similar structural contribution of the three products in secondary structure (Fig. 2C). The DSC thermograms of SB15 were comparable to US/EU-aflibercept and there were no meaningful differences in the measured melting points. Thus, it was demonstrated that the thermal transition and thermal stability of SB15 were similar to those of US/EU-aflibercept (Fig. 2D).

In addition, tertiary structure and conformational dynamics were characterized using H/DX-MS. The dynamics of deuterium uptake over time (10 s to 4 h) showed symmetry between compared samples (Fig. 3A, B) and no meaningful difference were observed in the individual difference and the sum of differences (Fig. 3C, D).

Fig. 3
figure 3

A–D Comparison for dynamic deuterium uptake of SB15 and US/EU-aflibercept. A Butterfly plot of SB15 and US-aflibercept, B butterfly plot of SB15 and EU-aflibercept, C difference plot of SB15 and US-aflibercept, D difference plot of SB15 and EU-aflibercept

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Purity and Impurity

The purity and impurity in terms of size heterogeneity were analyzed. The SE-HPLC chromatograms of SB15 and US/EU-aflibercept are presented in Fig. 4A. The samples were separated into an intense main peak (denoted as monomer) and a high molecular weight impurity peak (denoted as HMW). The relative quantity of HMW (%HMW) of SB15 (0.9%) were lower than US/EU-aflibercept (2.4 and 2.5%, respectively). The HMW impurity was confirmed as dimeric aggregates of aflibercept by SEC-MALS analysis (data not shown) and the aggregates of aflibercept can be increased during storage. The observed difference in %HMW has no biological impact (Table 3).

Fig. 4
figure 4

A–D Comparison for physicochemical properties of SB15 and US/EU-aflibercept A SE-HPLC chromatograms, B non-reduced CE-SDS electropherograms, C reduced CE-SDS electropherograms, D icIEF electropherograms. CE-SDS capillary electrophoresis-sodium dodecyl sulfate, HMW high molecular weight, icIEF imaging capillary isoelectric focusing, SE-HPLC size exclusion-high-performance liquid chromatography, STD standard, LMW low molecular weight

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In non-reduced CE-SDS, samples were separated into an intense main peak (denoted as main) and two impurity peaks related to low molecular weight (LMW) fragment (Fig. 4B). For all three products, %main were comparably high (99.6–99.7%). Under reduced condition, two main peaks (denoted as main 1 and main 2) and LMW impurity peaks were observed (Fig. 4C). The main 1 and main 2 peaks were related to partially N-glycosylated single chain and fully N-glycosylated single chain, respectively. The level of main 1 (%main 1), main 2 (%main 2), and LMW (%LMW) of SB15 were similar to those of US/EU-aflibercept.

Charge Heterogeneity

The charge heterogeneity of SB15 and US/EU-aflibercept were compared using icIEF method. Since aflibercept (SB15 and RP) is a highly glycosylated protein with terminal sialic acid on VEGFR-1 and VEGFR-2 domain, the charge profile of aflibercept (SB15 and RP) is highly complex. Thus, sialic acids were removed prior to icIEF analysis to efficiently assess the individual charge variant level and the sialic acid contents were measured using the orthogonal method.

In the electropherogram of the samples (Fig. 4D), the most intense single peak was denoted as main. The multiple peaks with lower pI value than main were assigned as acidic and the higher pI peaks were assigned as basic. The acidic levels (%acidic) of SB15 (67.1%) were lower than US/EU-aflibercept (75.5 and 75.6%) and the main levels (%main) were opposite. These observed differences in acidic and main level were mainly related to the level of deamidation including isoaspartate formation (data not shown). Also, the basic levels (%basic) of SB15 were lower than that of US/EU-aflibercept connected to different C-terminal variant levels. Although, a difference in charge heterogeneity was observed, it was not linked to changes in biological activities (Table 3).

N-Glycan Profile

The N-glycan species on SB15 and US/EU-aflibercept were identified using LC–ESI–MS. From the three products, 34 N-glycan species were identified and there was no N-glycan species detected in only SB15 nor US/EU-aflibercept (data not shown).

In the quantitation analysis, N-glycan species were categorized into 4 N-glycan groups (galactosylated glycan, afucose, charged glycan, and high mannose). As presented in Table 4, slight differences were observed in galactosylated and afucose level between SB15 and US/EU-aflibercept. However, aflibercept has no Fc-effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in its MoA. In addition, pharmacokinetics (PK) profile of SB15 was comparable to that of its RP [16] and no meaningful difference was observed in biological activities (Table 3).

Table 4 Summarized results of physicochemical quality attributes
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Biological Activity

The analytical similarity in terms of biological activity between SB15 and US/EU-aflibercept RP was demonstrated by MoA-related bioassays, including VEGF-A 165 binding, VEGF-A 121 binding, HUVEC anti-proliferation, and VEGF-A 165 neutralization assay, and Fc-related bioassay such as FcRn binding assay (Table 3). For the similarity assessment of biological activity, similarity ranges were established as mean ± kSD. In addition, binding assays to VEGF family ligands and Fc gamma receptors between SB15 and US/EU-aflibercept were performed. The results are provided in Figs. 5 and 6.

Fig. 5
figure 5

A–E Comparison for biological activities of SB15 and US/EU-aflibercept. Similarity ranges are presented bya dotted line. A VEGF-A 165 binding, B VEGF-A 121 binding, C HUVEC anti-proliferation, D VEGF-A 165 neutralization E FcRn binding. FcRn neonatal fragment crystallizable receptor, HUVEC human umbilical vein endothelial cells, PIGF placenta growth factor, VEGF vascular endothelial growth factor

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Fig. 6
figure 6

A–D Comparison for MoA-related additional biological activities of SB15 and US/EU-aflibercept. A VEGF-A 189 binding, B VEGF-B 167 binding, C PIGF-1 binding, D PIGF-2 binding. FcRn neonatal fragment crystallizable receptor, HUVEC human umbilical vein endothelial cells, PIGF placenta growth factor, VEGF vascular endothelial growth factor

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MoA-Related Biological Activity

MoA-related bioassays were conducted including VEGF-A 165 binding, VEGF-A 121 binding, HUVEC anti-proliferation potency, and VEGF-A 165 neutralization potency.

First, the evaluation of a relative VEGF-A 165 binding activity showed that the average of relative VEGF-A 165 binding activities of SB15 and US/EU-aflibercept were 104, 97, and 98%, respectively. There was no meaningful difference between SB15 and US/EU-aflibercept in VEGF-A 165 binding activities for all SB15 tested samples which were within the US/EU similarity range (Fig. 5A).

Second, the average of VEGF-A 121 binding activities of SB15 and US/EU-aflibercept were 100, 96, and 97%, respectively. There was no meaningful difference between SB15 and US/EU-aflibercept and VEGF-A 121 binding activities for all SB15 tested samples which were within the US/EU similarity range (Fig. 5B).

Third, the average of HUVEC anti-proliferation potency of SB15 and US/EU-aflibercept was 102, 97, and 99%, respectively. There was no meaningful difference between SB15 and US/EU-aflibercept and HUVEC anti-proliferation potencies for all SB15 tested samples which were within the US/EU similarity range (Fig. 5C).

Finally, the average of VEGF-A 165 neutralization potency of SB15 and US/EU-aflibercept was 101, 98, and 99%, respectively. There was no meaningful difference between SB15 and US/EU-aflibercept and VEGF-A 165 neutralization potencies for all SB15 tested samples which were within the US/EU similarity range (Fig. 5D).

Fc-Related Biological Activity

Fc-related bioassay was conducted to evaluate FcRn binding affinity, using SPR. The average of FcRn binding affinity of SB15 and US/EU-aflibercept was 99, 100, and 100%, respectively.

Relative FcRn binding affinities for all SB15 tested samples which were within the US/EU similarity range (Fig. 5E).

Additional Biological Activity

Aflibercept is a fusion protein of human VEGF receptor 1 and 2 extracellular domains with an Fc fragment of IgG1 and it can bind to VEGF-A, VEGF-B, and PlGFs.

SPR and ELISA methods were employed in order to assess binding activities of aflibercept (SB15 and RP) to VEGF family ligands such as VEGF-A 189, VEGF-B 167, and PlGFs.

Consequently, there were no meaningful differences between SB15 and US/EU-aflibercept in terms of VEGF family ligands binding activities (VEGF-A 189, VEGF-B 167, PIGF-1, and PlGF-2), respectively (Fig. 6).

Discussion

SB15 was developed by Samsung Bioepis as a biosimilar of reference aflibercept (Eylea®). In accordance with the guidelines, a high level of similarity of SB15 to aflibercept RP must be demonstrated in terms of structural and functional properties as well as their clinical outcomes.

Comprehensive characterization was implemented according to the guidance of FDA and EMA to demonstrate the analytical similarity of SB15 to RP using a panel of the state-of-the-art methods in terms of structural, physicochemical, and biological properties. Quality attributes for characterization were defined and ranked (“Tiered”) based on quality attribute risk assessment considering the potential impacts on PK/pharmacodynamics (PD), efficacy, safety, and immunogenicity [15]. Each quality attribute was evaluated for a demonstration of the analytical similarity through quality range approach, which was established from multiple batches of US/EU-aflibercept, or a side-by-side comparison.

The similarity in primary structure of SB15 to US/EU-aflibercept were confirmed as comparable in both peptide and protein level. In addition, the secondary and tertiary structures between SB15 and US/EU-aflibercept were demonstrated to be similar. The observed difference in PTM levels such as oxidation and deamidation were considered to be negligible in terms of biological activity. The purity and impurity in size heterogeneity of SB15 were similar to US/EU-aflibercept. There was an observed quality gap in charge heterogeneity between SB15 and US/EU-aflibercept. It was mainly related to deamidation and there was no associated biological impact. The N-glycosylation level of SB15 was slightly different than US/EU-aflibercept. However, it was deemed to be negligible because aflibercept has no Fc effector function in MoA and the PK of SB15 was comparable to that of its RP.

All biological characterization results of SB15 were within US and EU similarity ranges in terms of MoA-related biological activities, including VEGF-A 165 binding, VEGF-A 121 binding, HUVEC anti-proliferation, and VEGF-A 165 neutralization, and in terms of Fc-related biological activity such as FcRn binding. In additional biological assays, there were no meaningful differences between SB15 and US/EU-aflibercept in terms of VEGF family ligand binding (VEGF-A 189, PlGF-1, PlGF-2, and VEGF-B 167). Consequently, it was demonstrated that SB15 is similar to US/EU-aflibercept in terms of MoA-related and Fc-related biological activities.

In this study, only the reference aflibercept products (Eylea®) with expiry date between July 2016 and November 2022 were used for the analytical similarity assessment. However, the post-study monitoring revealed no meaningful quality changes, validating representativeness of the reference aflibercept products used in this study. In addition, our study was focused on the demonstration of analytical similarity between SB15 and its RP in terms of structural and functional properties. Thus, the detailed results of the following phase 3 clinical study were not discussed. Briefly, the randomized phase 3 clinical study was registered on ClinicalTrials.gov (SB15-3001; ClinicalTrials.gov: NCT04450329) and clinical comparability in efficacy, safety, PK/PD, and immunogenicity of SB15 to RP in participants with nAMD were demonstrated [16, 17]. Collectively, these findings provide evidence to support safe and effective use of SB15 as a biosimilar product of reference aflibercept.

Conclusions

The structural, physicochemical, and biological similarity between SB15 and US/EU-aflibercept were evaluated. Comprehensive analytical characterization of SB15 and US/EU-aflibercept were implemented and the results demonstrated that SB15 and US/EU-aflibercept were highly similar. The results of the analytical characterization provide the foundation for the totality of evidence to support the biosimilarity of SB15 with respect to US/EU-aflibercept.