Abstract
Background and Objective
Tocilizumab is an immunoglobulin G1 monoclonal antibody targeting the interleukin-6 receptor (IL-6R). BAT1806/BIIB800 (tocilizumab-bavi) has been developed as a biosimilar to the reference product tocilizumab (TCZ). The objective of this study was to demonstrate physicochemical and functional similarity between BAT1806/BIIB800 and TCZ in a comprehensive comparability exercise.
Methods
A comprehensive panel of over 20 methods was used to generate datasets comparing critical and non-critical product quality attributes for 10 BAT1806/BIIB800 lots and 44 TCZ lots (16 sourced from China, 16 from the EU, and 12 from the US). Primary structure, higher-order structure, and physicochemical properties were assessed using liquid chromatography, mass spectrometry, various spectroscopy techniques/methods, capillary electrophoresis, and thermoanalytical techniques. Fragment antigen-binding (Fab)- and fragment crystallizable (Fc)-mediated biological properties were assessed using cell-based assays, immunoassays, flow cytometry, and kinetic binding assays.
Results
BAT1806/BIIB800 and TCZ (irrespective of source) were shown to be similar in terms of structural and functional properties. No differences were observed in terms of the most critical quality attributes, that is, soluble-IL-6R binding and inhibition of IL-6-mediated cell proliferation. BAT1806/BIIB800 and TCZ demonstrated similarity in terms of Fab- and Fc-mediated binding and biological activity. Minor differences were observed in glycosylation (afucosylation and sialylation), glycation, aggregation, and charge variants, which were demonstrated to be not clinically relevant.
Conclusion
BAT1806/BIIB800 and TCZ were highly similar for all critical quality attributes. Where differences were observed in less critical quality attributes, additional analytical assessments and clinical study results determined these to be not clinically meaningful.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
A comparative analytical assessment of the biosimilar BAT1806/BIIB800 to reference tocilizumab was performed to assess structural, physicochemical, and functional quality attributes, essential for mechanism of action, and clinical safety and efficacy. |
Comprehensive comparative analytical assessments demonstrate BAT1806/BIIB800 similarity to reference tocilizumab in terms of critical quality attributes. Minor differences observed were shown to have no relevant clinical impact by additional analytical assessments and clinical studies. |
1 Introduction
BAT1806/BIIB800 (tocilizumab-bavi) has been developed as a biosimilar of reference tocilizumab (TCZ; marketed as RoActemra®/Actemra®). BAT1806/BIIB800 is produced in Chinese hamster ovary cells and consists of two kappa light chains (each containing 214 amino acid residues) and two immunoglobulin G (IgG)1 heavy chains (each containing 448 amino acid residues). Tocilizumab is a humanized interleukin-6 receptor (IL-6R) monoclonal antibody that binds to the soluble and membrane-bound IL-6 receptor variants (sIL-6R and mIL-6R), competitively inhibiting IL-6 signalling [1, 2]. Tocilizumab has been approved for the treatment of inflammatory conditions, including moderate-to-severe rheumatoid arthritis (RA), adult giant cell arteritis, polyarticular juvenile idiopathic arthritis or systemic juvenile idiopathic arthritis, and is being used for the treatment of patients with COVID-19 pneumonia [3, 4]. BAT1806/BIIB800 was recently approved by the China National Medical Products Administration (NMPA) and the US Food and Drug Administration (FDA) for marketing authorization under the trademark name Tofidence™ in the USA.
Regulatory guidelines require that biosimilars demonstrate a high degree of similarity with the reference product(s) (RP), in terms of structural, physicochemical, and biological properties (including potency), as well as general properties such as protein content and protein concentration, recommending a stepwise, totality-of-evidence approach [5,6,7,8,9]. In general, a biosimilar is expected to have the same strength in terms of protein concentration as the RP although in some jurisdictions, including Europe, it is permitted to introduce new strengths, if properly justified [9, 10]. The stepwise approach initially entails conducting a comprehensive structural analysis to gain an in-depth understanding of the RP profile to determine the quality target product profile. Once the quality target product profile of the RP is established, a novel manufacturing process to produce a biosimilar candidate is developed. As the manufacturing process of the RP is proprietary, a manufacturer of biosimilars will need to develop its own cell lines and manufacturing methods. Given these manufacturing differences, and the inherent variability of the manufacturing process of biologics, biosimilars are expected to have analytical differences when compared with the RP. Any differences observed between a proposed biosimilar and its RP must be justified as not being clinically meaningful with respect to efficacy and safety, and may require providing additional data from analytical, functional, and/or clinical studies [5,6,7, 11].
Clinical similarity of BAT1806/BIIB800 compared with TCZ was demonstrated in published phase I and III studies [12, 13]. Here we present the physicochemical, structural and biological characterization of BAT1806/BIIB800 using a comprehensive comparability exercise applying a multitude of analytical techniques and in vitro assays to determine its similarity with China-, EU- and US-sourced TCZ.
2 Materials and Methods
The similarity testing plan is summarized in Supplementary Table 1. A comprehensive panel of over 20 methods was used to generate a detailed, physicochemical, structural and functional comparison of critical and non-critical product quality attributes for 10 lots of BAT1806/BIIB800 and 44 lots of TCZ (16 sourced from China [CN], 16 from the European Union [EU], and 12 from the United States [US]).
BAT1806/BIIB800 is formulated in His buffer 10 mM (containing histidine and histidine monohydrochloride monohydrate), arginine-hydrochloride (HCl) 50 mM, sucrose 20 mg/mL, polysorbate 80 (pH 6.2) 0.5 mg/mL, while TCZ is formulated in phosphate buffer15 mM, sucrose 50 mg/mL, polysorbate 80 (pH 6.5) 0.5 mg/mL.
Analytical similarity acceptance criteria were established based on the criticality of the product quality attributes, method capability, and lot-to-lot variability of the RP. A Tier system (as shown in Supplementary Table 2, see electronic supplementary material [ESM]) was used to rank and classify each quality attribute and associated methods in accordance with regulatory guidance [14, 15]. The attributes that directly relate to the primary mechanism of action are classified as the most critical quality attributes (CQAs) and are designated as Tier 1 (high clinical impact). For product quality attributes placed in Tier 1, an equivalence test was used where the proposed biosimilar is considered equivalent to the RP if the calculated 90% two-sided confidence interval of the mean difference between the proposed biosimilar product and RP is within ± δ. The δ was based on the standard deviation of all the RP lots tested and multiplied by 1.5. Tier 2 classification comprised quantitative assays measuring high-impact CQAs by orthogonal methods. The most sensitive method was used for quantitative characterization test results. Tier 2 group similarity acceptance criteria were categorized into analysis by quality range (QR). The similarity acceptance criterion for a Tier 2 QR-based analysis was > 90% of BAT1806/BIIB800 lots that were to be within the RP mean ± 3 standard deviations. Tier 3 classification covered quantitative assays measuring non-CQAs (with lower clinical risk) and semi-quantitative assays or assays producing a fingerprint or non-quantitative output (e.g., a spectra or chromatographic comparison). For Tier 3 attributes, the similarity assessments used raw data/graphical comparisons.
All analyses were performed at Bio-Thera Solutions, Ltd with the exception of far ultraviolet (UV) and near UV by circular dichroism, performed at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, and sedimentation coefficient by sedimentation velocity analytical ultracentrifugation (SV-AUC), performed by Shanghai ZhenGe Biotech Co. Ltd. Samples were analyzed under identical conditions for both BAT1806/BIIB800 and TCZ.
2.1 Materials
The BAT1806/BIIB800 drug product was manufactured by Bio-Thera Solutions Ltd (Guangzhou, China), while the lots of TCZ were purchased from the Chinese (CN source), EU (EU source), and US markets (US source), and stored according to the manufacturer’s instructions.
2.2 Analytical Methods
2.2.1 Intact and Reduced Deglycosylated Mass Analysis by Liquid Chromatography–Mass Spectrometry (LC–MS)
Intact mass analysis was performed by initial desalting using ultracentrifugation (Millipore, Amicon Ultra Centrifugal Filter, 3 kDa MWCO) followed by reverse phase (RPh) LC chromatographic resolution with a BioResolve™ RP (450 Å, 2.1 × 100 mm, 2.7 μm) column (Waters, Milford, MA, USA) on a Waters ACQUITY Bio H-Class ultra performance liquid chromatography (UPLC) system (Waters). Analysis was performed using UV adsorption (ACQUITY UPLC® Tunable UV Detector) and electrospray high-resolution accurate mass spectrometry (HRMS) on a Vion IMS QTof (Waters) using UNIFI software (Waters, v 1.8.2.16).
To determine the similarity of the antibodies at the heavy chain (HC) and light chain subunit level, samples were denatured with 6 M guanidine HCL (Aladdin) and reduced with 0.5 M dithiothreitol (DTT, Sigma) to generate reduced samples. For deglycosylated analysis, samples were diluted with Tris-HCl (pH 7.95, Aladdin) and treated with PNGase F (New England Biolabs); for reduced and deglycosylated analysis, intact antibodies were treated with PNGase F then denatured and reduced as before.
The intact mass analysis was summed, and the deconvoluted spectrum of BAT1806/BIIB800 (intact, reduced, and deglycosylated) was compared with lots of TCZ.
2.2.2 Reduced and Non-reduced Peptide Mapping by LC–MS
Reduced peptide mapping was performed by denaturation of samples with 6 M guanidine HCl (Aladdin) and DTT in a 100-mM Tris buffer. Iodoacetamide (0.5 M, Sigma) was used to cap the reduced thiols and the samples were then digested with trypsin (Promega) or Asp-N (Promega).
Non-reduced peptide mapping was performed by denaturation of test samples with 6M guanidine HCl (Aladdin) followed by digestion with either trypsin (Promega) or Asp-N (Promega) in the same manner as for reduced analysis.
Digested samples were chromatographically resolved using an ACQUITY UPLC® Peptide BEH C18 Column (300 Å, 1.7 µm, 2.1 mm × 150 mm, Waters) on an ACQUITY UPLC H-Class system (Waters). Samples were detected by UV absorbance at 214 nm and electrospray HRMS on Xevo G2-S QTof (Waters). Data collection was performed using MassLynx v4.1 software and processed with BiopharmaLynx v1.3.3 software (Waters) with disulphide bond positions identified by comparison of data generated under reducing and non-reducing conditions.
2.2.3 Glycan Mapping by Hydrophilic Interaction Chromatography (HILIC)-High-Performance Liquid Chromatography (HPLC)
Samples were denatured in RapiGest™ (Waters), reduced with DTT then treated with glycopeptidase F (Takara). The glycans were precipitated with acetonitrile and the supernatant was dried. Isolated N-glycans were labelled with procainamide HCl (Sigma) and chromatographically resolved using a HILIC XBridge BEH Amide (2.5 μm 3.0 × 100 mm) column (Waters) on an ACQUITY Arc HPLC (Waters) with fluorescent (FLR) detection (2475FLR, Waters) at an excitation wavelength of 310 nm and an emission wavelength of 370 nm. Data were acquired and processed by the integration algorithm Empower™3 (Waters) software and results reported as relative percentage of glycan (e.g., glycoform percent G0F, percent G1F, and percent G2F).
2.2.4 Total Sialic Acids by RPh HPLC-FLR
N-Acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) content were evaluated by hydrolyzing the samples and the NANA and NGNA standard curves (Sigma), and the isolated glycan samples with 4M acetic acid. Samples were chromatographically resolved using a Kromasil 100-5-C18 column (4.6 × 250 mm, 5 µm) on an ACQUITY Arc HPLC instrument (Waters) with FLR detection (2475FLR, Waters) at an excitation wavelength of 373 nm and an emission wavelength of 448 nm. Data based on NANA and NGNA calibration curves were acquired and processed by Empower™3 (Waters), and results reported as molar total sialic acid amount per mole of antibody.
2.2.5 Free Thiol Content by Ellman Method
Free thiol content was evaluated by treating test samples and a calibration line of L-Cys (Sigma) with Ellman's reagent (5,5′-dithiobis-[2-nitrobenzoic acid]). After the reaction, samples were analyzed on a UV-2600 UV spectrophotometer (Shimadzu) at 412 nm. Free thiol concentrations of test samples were calculated from the L-Cys calibration line.
2.2.6 Capillary Isoelectric Focusing
Test samples were mixed with Pharmalyte (Cytiva), isoelectric point (pI) markers (BioCEart, Beijing Biosmart Technical Institute), 4 M urea-capillary isoelectric focusing gel (Aladdin), 0.5 M arginine (Aladdin), and 0.2 M N-2-acetamido iminodiacetic acid (Aladdin). Each mixture was resolved using a μsilFC coated capillary (BioCEart) on a CE 7100 capillary electrophoresis system (Agilent). Data were acquired and processed by OpenLAB software (Agilent).
2.2.7 Extinction Coefficient
Extinction coefficient was calculated using the Edelhoch method [16].
2.2.8 Circular Dichroism Spectroscopy
Formulation buffer was removed from the test samples by ultrafiltration (12,000 rpm) and samples were diluted with phosphate buffer 5 mM (Sigma, 10 and 0.1 mg/mL). Secondary and tertiary structures were assessed with 10 mg/mL for near and 0.1 mg/mL for far UV-circular dichroism (UV-CD), respectively, using Chirascan V100 spectrophotometer UV-CD (Applied Photophysics Ltd) controlled by Chirascan software (Applied Photophysics Ltd). The CD absorption spectra in the far- and near-UV regions were measured at 190–260 nm (to determine secondary structures) and 250–350 nm (to determine tertiary structures), respectively. After scanning and data collection, Chirascan software was used to obtain averaged, baseline subtracted, and smoothed replicates of three spectra per sample.
2.2.9 Fourier-Transform Infrared Spectroscopy
Secondary structure was determined using spectral scanning of freeze-dried, powdered samples on an attenuated total reflection Fourier-transform infrared spectrometer (Thermo Fisher Scientific Inc., Nicolet iS10), running OMNIC Spectra software (Thermo Fisher Scientific Inc.). After background removal and second derivative processing (smoothed twice at level 9), spectra for test samples were compared.
2.2.10 Endogenous Fluorescence
The tertiary structure of test samples was compared by endogenous fluorescence using an Uncle-0383 static light scattering charge-coupled device FLR detector (Unchained Labs) scanning from 250 to 450 nm. After data collection, spectra were analyzed using Uncle Analysis software (Unchained Labs). Comparison of data was performed by visual comparisons.
2.2.11 Differential Scanning Calorimetry (DSC)
DSC was used to measure the thermal unfolding temperatures of specific domains of test samples on a MicroCal VP-DSC (Malvern Panalytical). Samples were diluted to 2 mg/mL with preparation buffer and heated from 40 to 120 °C (heating rate of 110 °C/h) in triplicate. Software was used to process and analyze melting temperature (MicroCal VP-Capillary DSC software, Malvern Panalytical) and normalization for protein concentration; the melting temperature was determined using MicroCal Analysis Launcher software (Malvern Panalytical).
2.2.12 Subvisible Particles by Light Obscuration and FlowCam
Subvisible particles of test samples were assessed by light obscuration using a HIAC 9703+ liquid particle counting system (Beckman Coulter) controlled by PharmSpec software (Beckman Coulter). Particle concentration results were reported as cumulative particle counts per mL for ≥ 2, ≥ 5, ≥ 10, ≥ 25, and ≥ 50 µm size ranges. Samples were also assessed using the FlowCam 8000 (Yokogawa Fluid Imaging Technologies, Inc.) particle imaging system containing a flow cell and a digital camera. Spherical and non-spherical particle counts per mL for particles ≥ 5 µm were reported as those product-related particles that were more likely to be proteinaceous, and potentially have an increased risk to illicit an immunogenic response.
2.2.13 Submicron Particle Size Concentrations by Dynamic Laser Light Scattering (DLS)
The translational diffusion coefficient for particles in solution was determined by DLS using a DynaPro NanoStar DLS (Waters). Samples were prepared at 25 °C and the determined coefficients were used to calculate the equivalent spherical hydrodynamic radius. DLS profiles were performed by visual comparisons.
2.2.14 Size Exclusion Chromatography (SEC) With Multi-Angle Light Scattering (MALS)
SEC with MALS was used to assess the similarity between test samples for aggregates. Samples were chromatographically resolved using a TSK-gel G3000SWXL analytical column (5 µm/7.8 mm × 300 mm, Tosoh) on an e2695 HPLC system (Waters) with a mini-DAWN MALS detector (Wyatt) and a W2414 refractive index detector (Waters). Data were collected and processed using ASTRA software (Wyatt) to generate the absolute molecular weight of monomer and polymer species in kDa.
2.2.15 Aggregate Profile by SV-AUC
An AUC consists of an optical detection system integrated into an ultracentrifuge allowing for real-time detection of the evolution of the concentration distribution of particles subjected to centrifugation. Aggregate profiles of test samples were determined using SV-AUC and performed on an Optima AUC analytical ultracentrifuge (Beckman Coulter) at 4000 rpm and the detection wavelength of 280 nm. AUC-SV was used to characterize aggregates and fragments with respect to heterogeneity and particle size. Samples were analyzed in triplicate and process by SEDFIT software (https://sedfitsedphat.nibib.nih.gov/software/default.aspx).
2.2.16 Size Variants by SEC-HPLC
Size variants of each sample were determined by SEC-HPLC with UV absorbance. Chromatographic resolution was achieved with a Tosoh Bioscience TSK Gel G3000 SWXL column (7.8 × 300 mm, 5 µm, Tosoh) on an e2695 HPLC system (Waters) using a 2489 UV/Vis Detector scanning at 280 nm. Data were acquired and processed by Empower™3 (Waters) software.
2.2.17 Low Molecular Weight Fragments by Capillary Electrophoresis (CE)-Sodium Dodecyl Sulfate (SDS)
Low molecular weight fragments present in samples were determined using CE-SDS either on reduced or non-reduced samples. For reduced analysis, samples were mixed with 70 µL of CE-SDS sample buffer (pH 9.4, BioCEart), and 5 µL of 2-Mercaptoethanol (Sigma) and boiled at 70 °C for 10 min. For non-reduced analysis, samples were mixed with 70 µL of CE-SDS sample buffer (pH 6.5) and 5 µL of iodoacetamide (Sigma) and heated at 65 °C for 4 min. Both sets of samples were electrophoretically separated using a bare fused-silica capillary (50 µm, 33 cm, Beckman Coulter) on an Agilent CE 7100 (Agilent Technologies) using UV detection at 220 nm. Data were acquired and processed by OpenLAB software with integration capabilities (Agilent).
2.2.18 Charge Variants by Ion Exchange (IEC)-HPLC
IEC-HPLC was used for the assessment of charge variants of test samples with and without carboxypeptidase B (CpB) treatment. For CpB treatment, samples were diluted with 20 mM of ACES buffer (pH 7.5) to a concentration of 5 mg/mL, then CpB (YAXINBIO, CAS #9025-24-5) at 5 mg/mL was added at a 50:1 (sample:CpB) ratio, mixed and incubated (30 min, 37 °C). Both sets of samples were then chromatographically separated on a Thermo MAbPac SCX-10 (10 μm, 4.0 × 250 mm) column (Thermo Fisher Scientific) using a 1260 HPLC system (Agilent) equipped with an e2695 UV detector (Waters). Data were acquired and processed by Empower™3 (Waters) software, using the ApexTrack integration algorithm. Results were reported as a relative percentage of the charge variants (e.g., percent of acidic region, percent of main peak, and percent of basic region).
2.2.19 Purity Hydrophobic Interaction (HIC)-HPLC and RPh-UPLC
HIC-HPLC was used to separate intact antibodies from their aggregates and variants under non-denaturing conditions. Chromatographic separation was performed with an Agilent Bio HIC (4.6 × 100 mm, 3.5 µm) column (Agilent) on an e2695 HPLC system (Waters) equipped with a 2489 UV/Vis Detector and data were collected at 280 nm. Data collection was performed using Empower™3 (Waters) software with the percentage of main peak, percentage of pre-peaks (eluting earlier that main peak), and percentage of post-peaks (eluting after main peak) evaluated.
RPh-UPLC was used to compare different proteins by their relative hydrophobicity. RPh is more denaturing than HIC and therefore offers complementary analysis. Chromatographic separation was performed on a BioResolve RPh mAb Polyphenyl (450 Å, 2.7 μm, 2.1 mm × 100 mm) column (Waters) and an ACQUITY UPLC H-Class Bio UPLC system (Waters) equipped with a Tunable UV detector (Waters), collecting data at 280 nm. Data collection was performed as described for HIC-HPLC.
2.3 Functional Assays
In addition to structural and physicochemical characterization, functional similarity is an essential part of biosimilarity. Given that the mechanism of action of tocilizumab is the inhibition of IL-6 binding to both sIL-6R and mIL-6R and, therefore, receptor signalling, multiple assays targeting this pathway were used to demonstrate that BAT1806/BIIB800 is similar to TCZ in terms of biological activity.
2.3.1 Binding to sIL-6R by Enzyme-Linked Immunosorbent Assay (ELISA)
Direct binding of test samples to sIL-6R was determined via an ELISA where recombinant human sIL-6R (Sino Biological) was coated onto ELISA plates. In brief, serially diluted samples were added to the sIL-6R immobilized plate, washed, then detected by goat anti-human IgG (fragment crystallisable [Fc] fragment) conjugated to horseradish peroxidase (HRP, Jackson ImmunoResearch) followed by 3,3′,5,5′ tetramethylbenzidine (TMB; Sigma-Aldrich) substrate. The reaction was terminated with 0.2 M sulfuric acid and absorbance measured with a SpectraMax M4 microplate reader (Molecular Devices) at 450 nm. After assessing parallelism of the dose–response curves, the binding activity of test samples relative to the qualified BAT1806/BIIB800 internal reference standard, B0520180301STD, derived from the commercial process lot (B0520180301; referred to as ‘reference standard’ hereafter) was determined using a four-parameter logistic model fit by SoftMax Pro Software (Molecular Devices). Results were reported as a percentage of the relative binding values.
2.3.2 Competitive Inhibition of IL-6 Binding to sIL-6R by ELISA
Competitive inhibition of test samples was determined by immobilization of recombinant human sIL-6 (Bio-Thera Solutions) onto ELISA plates. Serial dilutions of test samples and reference standard were mixed with IL-6R-His (Bio-Thera Solutions) at a fixed concentration of 1 µg/mL. After washing, rabbit anti-His antibody (Bio-Thera Solutions) was added, followed by anti-rabbit IgG conjugated to HRP (Wuhan Boster Biological Technology Co., Ltd.) and TMB (Sigma-Aldrich) substrate. The reaction was terminated with 0.2 M sulfuric acid, absorbance measured, and the binding activity of samples relative to the reference standard was determined as before. Results were reported as a percentage of the relative binding value.
2.3.3 Binding Kinetics to sIL-6R by Surface Plasmon Resonance (SPR)
Binding kinetics of test samples to sIL-6R (Sino Biological) were assessed by SPR using a Biacore™ T200 (GE HealthCare) running multiple-cycle kinetics. sIL-6R serial concentrations were added to a carboxy-methylated sensor chip (GE HealthCare) with pre-immobilized Protein A, which was used as the Fc region capture reagent of each sample. On-rates (ka), off-rates (kd), and equilibrium binding were calculated. Data collection was performed by Biacore T200 Evaluation software (GE HealthCare).
2.3.4 Inhibition of Signal Transducer and Activator of Transcription (STAT) 3 Phosphorylation by Homogeneous Time Resolved Fluorescence (HTRF)
IL-6 binding to mIL-6R results in a complex that recruits glycoprotein (gp)130 to activate intracellular signalling [17]. gp130 dimerization activates Janus kinase (JAK), a tyrosine receptor kinase, which phosphorylates tyrosine residues on gp130 to recruit and phosphorylate signal transducers and STAT3 [17]. Phosphorylated STAT3 then relocates into the nucleus where it regulates transcription and expression of target genes, activating a variety of immune responses [18]. The ability of test samples to inhibit STAT3 phosphorylation was determined by an HTRF assay (Cisbio, 62AT3PEG) on a SpectraMax M5e FLR microplate reader (Molecular Devices). TF-1 cells (human erythroleukemia cell line expressing mIL-6R) were incubated with serial test sample concentrations, then stimulated with a constant concentration of IL-6 (Sino Biological, 8 ng/mL). Following lysis, phosphorylated STAT3 was detected using the Phospho-STAT3 (Tyr705) kit (Cisbio) and a SpectraMax M5e FLR microplate reader (Molecular Devices) equipped with the HTRF detection model. The percentage of the relative potency of samples was calculated against a dose–response curve of the reference standard. Data collection and processing was achieved by SoftMax Pro Software (Molecular Devices).
2.3.5 Inhibition of IL-6/sIL-6R–Induced Vascular Endothelial Growth Factor (VEGF) Release in Human Fibroblast-Like Synovial Cells (HFLS)
IL-6 and sIL-6R in the synovia of patients with RA have been shown to correlate with local joint inflammation and the severity of joint damage in chronic synovitis [19, 20]. VEGF is upregulated by proinflammatory cytokines, including IL-6 [21]. To determine the ability of test samples to inhibit VEGF secretion, a cell-based ELISA assay was developed using HFLS cells from patients with RA (HFLS-RA). HFLS cells, which do not express mIL-6R but do express gp130, respond to IL-6 and sIL-6R stimulation through the trans-signalling pathway. HFLS-RA cells (Cell Applications, Inc.) were incubated with serial test sample concentrations, the reference standard, and a fixed dose of IL-6 and sIL-6R (both from Sino Biological). After incubation, secreted VEGF was detected according to the manufacturers’ instructions (human VEGF ELISA kit, Wuhan Boster Biological Technology Co., Ltd.). The relative potency of test samples was calculated against the reference standard at each concentration. Data collection and processing was achieved by SoftMax Pro Software (Molecular Devices).
2.3.6 Binding to mIL-6R by Flow Cytometry
Direct binding of test samples to mIL-6R was performed by incubating various concentrations of samples and reference standard with mIL-6R-overexpressing HEK-Blue™ IL-6 cells (InvivoGen). After washing, Alexa Fluor 488 goat anti-human IgG (Thermo Fisher Scientific Inc.) was added and mean fluorescence of FITC-A detected by flow cytometry on a CytoFLEX (Beckman Coulter) was evaluated. CytExpert software (Beckman Coulter) was used for data collection. Dose–response curves of the reference standard and samples were fitted using a four-parameter logistic model by GraphPad Prism 8 (GraphPad) and the binding activity of samples relative to the reference standard was determined.
2.3.7 Inhibition of IL-6–Mediated Proliferation in TF-1 Cells
Inhibition of IL-6–mediated proliferation by test samples was determined using TF-1 cells engineered to express mIL-6R (National Institutes for Food and Drug Control China). TF-1 cells were incubated with varying concentrations of reference standard and test samples in the presence of IL-6 (4 ng/mL, Sino Biological). After incubation, CellTiter-Glo® Luminescent Cell Viability Assay (Promega) solution was added and luminescence measured with a SpectraMax M5e microplate reader (Molecular Devices). The percentage of the inhibition of TF-1 cell proliferation relative to the reference standard was determined using a four-parameter logistic model fit by SoftMax Pro Software (Molecular Devices).
2.3.8 IL-6 Blockage Activity by Secreted Embryonic Alkaline Phosphatase (SEAP) Reporter Gene Assay
The SEAP reporter gene assay was developed based on the JAK/STAT3 signalling pathway to evaluate the blocking activity of test samples against IL-6. HEK-Blue™ IL-6 cells expressing mIL-6R, STAT3 cDNA, and interferon-β response elements–driven SEAP reporter genes were incubated with varying concentrations of reference standard and test samples in the presence of IL-6 (1.5 ng/mL, Sino Biological). IL-6–mediated activation of STAT3 and downstream SEAP was measured by the addition of QUANTI-Blue™ (InvivoGen) solution and quantified with a SpectraMax M5e microplate reader (Molecular Devices). Blockage activity was calculated against a dose–response curve of the internal reference standard by SoftMax Pro Software (Molecular Devices).
2.3.9 Binding Affinity to Fc Receptors (neonatal fragment crystallisable receptor [FcRn] and fragment crystallisable gamma receptors [FcγR] IIa, IIb, IIIa) by Bio-Layer Interferometry (BLI)
The FcRn, FcγRIIa-131H/131R, FcγRIIb, FcγRIIIa-158V/158F binding affinity of test samples was determined by BLI using an Octet QKe platform (Sartorius) with multiple-cycle kinetics. Briefly, streptavidin sensors (Sartorius) were used to load the biotinylated FcRn (ACRO Biosystems), FcγRIIa-131H/131R (ACRO Biosystems), FcγRIIb (ACRO Biosystems), and FcγRIIIa-158V/158F (ACRO Biosystems), respectively. Reference standard and test samples were tested at varying concentrations with the affinity constant KD obtained using the 1:1 kinetic or steady-state fitting model. Data were collected and processed with Octet Data Analysis software (Sartorius). Results were reported as a percentage of affinity relative to reference standard.
2.3.10 Binding Affinity to Complement Component 1q (C1q) by BLI
To determine the ability of test samples to bind to C1q, BLI with multiple-cycle kinetics was performed on an Octet QKe platform using a sensor chip with pre-immobilized Protein L sensors (Sartorius) to capture samples via their light chain. The percentage of relative binding was reported by comparing the affinity constant KD (which was obtained by the 1:1 kinetic model fitting) from test samples against the reference standard. Data were collected and processed with Octet Data Analysis software (Sartorius).
2.3.11 Binding Affinity to FcγRIa by SPR
FcγRIa binding was assessed by SPR with multiple-cycle kinetics on a Biacore™ T200 (GE HealthCare), using a sensor chip with pre-immobilized Protein A (GE HealthCare). Reference standard and test samples were assessed at varying concentrations with the affinity constant KD obtained using the 1:1 kinetic or steady-state fitting model. Results were reported as a percentage of affinity relative to reference standard. Data were collected and processed with Biacore T200 Evaluation software (GE HealthCare).
2.3.12 Binding Affinity to FcγRIIIb by SPR
FcγRIIIb binding was assessed by SPR with multiple-cycle kinetics on a Biacore™ T200 (GE Healthcare) using a CM5 Chip (GE Healthcare) with pre-immobilized anti-His Ab (GE HealthCare) to capture human FcγRIIIb conjugated with His tag (R&D Systems). Samples were tested at various concentrations in parallel and reported as affinity constant KD by the steady-state model fitting. Data were collected and processed with Biacore T200 Evaluation software (GE HealthCare).
2.3.13 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
ADCC activity of test samples was evaluated by two reporter gene assays and one peripheral blood mononuclear cell (PBMC)-based lactate dehydrogenase (LDH) cytotoxicity assay. For the reporter gene assay, mIL-6R-expressing TF-1 and HEK-Blue™ IL-6 cells were used as target cells, and Jurkat cells overexpressing FcγRIIIa (158V) and nuclear factor of activated T cells (NFAT) response elements-driven luciferase (Jurkat/NFAT-luc+ FcRγRIIIa [158V]) were used as effector cells. For the PBMC-based LDH cytotoxicity assay, TF-1 cells and PBMC were used as the target and effector cells, respectively. Raji cells (CD20 expressing cell line) with ofatumumab (an anti-CD20 monoclonal antibody, Novartis) were used as a positive control, while pertuzumab (an anti-Her2 monoclonal antibody, Novartis) was a negative control. Briefly, target and effector cells were seeded in 96-well plates and incubated with a serial dilution of test samples. Luciferase or cell cytotoxicity were detected using a Bio-Lite™ Luciferase Assay System (Vazyme) or CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) and quantified with a SpectraMax M5e microplate reader (Molecular Devices). Data were collected and processed with SoftMax Pro software (Molecular Devices).
2.3.14 Complement-Dependent Cytotoxicity (CDC)
The CDC activity was evaluated using TF-1 and HEK-Blue™ IL-6 cells as target cells and human serum complement. Raji cells with ofatumumab were used as a positive control and pertuzumab as a negative control. Briefly, target cells were seeded in 96-well plates and incubated with a serial dilution of test samples, followed by human complement (Quidel). Cell viability was detected using Cell Counting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme) reagent and quantified with a SpectraMax M5e microplate reader (Molecular Devices). Data were collected and processed with SoftMax Pro software (Molecular Devices).
3 Results
The similarity testing plan to compare BAT1806/BIIB800 and TCZ (CN, EU and US) is summarized in Supplementary Table 1 (see ESM) and includes state-of-the-art, primary, and orthogonal methods to analyze quality attributes. Supplementary Table 2 summarizes the tier classification approach and associated acceptance criteria employed to assess the biosimilarity of BAT1806/BIIB800 with TCZ (see ESM). Table 1 shows the comparative analytical assessments for BAT1806/BIIB800 and TCZ. BAT1806/BIIB800 and TCZ were shown to be similar for all sources (CN, EU, and US), for all quality attributes, with the exception of glycosylation heterogeneity (percent mannose, percent sialylation, percent afucosylation, and percent galactosylation), aggregation, and charge variants, for which minor differences were observed.
3.1 Primary Structure
Similarity of the primary structure between BAT1806/BIIB800 and TCZ was assessed using several complementary characterization methods, including intact protein mass analysis, reduced and non-reduced peptide mapping, free-thiol determination, glycan mapping, extinction co-efficient, and isoelectric point analysis. For these analyses, BAT1806/BIIB800 was demonstrated to be similar to TCZ, with the exception of glycan composition (Fig. 1; Supplementary Fig. 1, Table 1). Differences in glycation content were identified in deglycosylated intact mass analysis (LC–MS).
Comparison for non-reduced (a) and reduced peptide (b) maps by tryptic digestion of BAT1806/BIIB800 and TCZ. BPI base peak intensity, CN China, EU European Union, H heavy, L light, TCZ tocilizumab, US United States
Glycan analysis was performed quantitatively by HILIC-HPLC analysis, with test sample chromatograms appearing visually similar to TCZ with no additional peaks present (Fig. 2). BAT1806/BIIB800 differed slightly in the percentage of composition of glycans compared with TCZ, with a minor increase in galactosylation, increased sialylation, decreased afucosylation, and reduced percentage of high mannose (Table 1). Notably, sialylation in both BAT1806/BIIB800 and TCZ was present at very low levels (Table 1; Fig. 2e). NANA was determined to be the predominant form of sialic acid in both BAT1806/BIIB800 and TCZ (Table 1).
Comparison for N-glycan of BAT1806/BIIB800 and TCZ. a N-Glycan profiles. b Scatter plot of galactosylation. c Scatter plot of high mannose. d Scatter plot of afucosylation. e Scatter plot of sialylation. CN China, EU European Union, TCZ tocilizumab, US United States
3.2 Higher Order Structure
Higher order structure of BAT1806/BIIB800 and TCZ was assessed using multiple biophysical techniques. Secondary structure was characterized by Fourier-transform infrared spectroscopy and far UV-CD, whilst tertiary structures were compared with near UV-CD, intrinsic fluorescence, and DSC. Higher order structure analyses by all methods demonstrated that BAT1806/BIIB800 is similar to TCZ (Supplementary Fig. 2 [see ESM], Table 1).
3.3 Particles and Aggregates
Particle content and aggregate profiles for BAT1806/BIIB800 and TCZ were assessed using a combination of methods that detect the presence of and/or quantify particles and aggregates of various sizes and shapes (spherical or non-spherical). Subvisible particles were determined by light obstruction, submicron particles were compared by DLS and SEC-MALS, and aggregate profiling was performed by SV-AUC. Particle content and aggregate analyses demonstrated that BAT1806/BIIB800 is similar to TCZ for most attributes (Table 1). Non-spherical particles ≥ 5 µm showed a wider range with BAT1806/BIIB800 compared with TCZ but were within the variability of the assay.
3.4 Product-Related Substances and Impurities
Size variants were assessed by SEC-HPLC, and reduced and non-reduced CE-SDS (Fig. 3). Charge profiles were compared by IEC-HPLC with and without CpB (Fig. 4). BAT1806/BIIB800 was shown to be similar to TCZ for most of the product-related substance and impurity analyses (Table 1). However, similarity assessment of monomer content and percentage of high molecular weight species (identified as dimers by SEC-MALS) in BAT1806/BIIB800 and TCZ samples showed that, for BAT1806/BIIB800, levels were below the lower limit of the quality range for the percentage of high molecular weight, while the monomer content was found to be above the upper limit of the quality range.
Comparison for size heterogeneity of BAT1806/BIIB800 and TCZ. a SEC-HPLC profile. b Scatter plot of HMW in SEC-HPLC. c nrCE-SDS profile. d Scatter plot of pre-peaks in nrCE-SDS. AU absorbance unit, C chain, CN China, EU European Union, H heavy, HMW high molecular weight, L light, LMW low molecular weight, m milli, nrCE-SDS non-reduced capillary electrophoresis-sodium dodecyl sulfate, SEC-HPLC-MALS, size exclusion high-performance liquid chromatography with light scattering, TCZ tocilizumab, US United States
Comparison for charge heterogeneity of BAT1806/BIIB800 and TCZ. a IEC-HPLC profile before CpB digestion. b Scatter plot of acidic region in IEC-HPLC before CpB digestion. c IEC-HPLC profile after CpB digestion. d Scatter plot of acidic region in IEC-HPLC after CpB digestion. AU absorbance units, CpB carboxypeptidase B, CN China, EU European Union, HPLC high-performance liquid chromatography, IEC cation exchange, TCZ tocilizumab, US United States
Similarity of charge profiles was determined by IEC-HPLC (with and without CpB) and quantitative analysis prior to CpB digestion confirmed that the acidic region, main peak, and basic region of BAT1806/BIIB800 were within the quality range of TCZ (Fig. 4, Table 1). Following CpB digestion, acidic and basic regions were shown to be similar to TCZ and within the quality ranges, with the caveats that the basic region was marginally lower in BAT1806/BIIB800 compared with TCZ and the main peak was slightly higher (Fig. 4, Table 1).
Protein variants were assessed for similarity by HIC-HPLC (a non-denaturing technique) and RPh-UPLC (a denaturing technique; complimentary to HIC-HPLC) (Table 1). Minor differences in the contents of the hydrophobic variants between BAT1806/BIIB800 and TCZ were observed, but these were within the quality range.
3.5 Functional Biological Activity
A comprehensive panel of biological assays was executed to compare Fab-mediated activity of BAT1806/BIIB800 and TCZ. These assays included direct binding to sIL-6R, competitive inhibition, and binding kinetics to sIL-6R, direct binding to mIL-6R, inhibition of IL-6-stimulated HEK-Blue™ IL-6 cells, inhibition of IL-6-mediated TF-1 cell proliferation, inhibition of STAT3 phosphorylation, and inhibition of VEGF release in HFLS-RA cells.
Direct binding to sIL-6R (Table 1, Fig. 5) demonstrated similarity between BAT1806/BIIB800 and TCZ with statistical equivalence testing confirming that the results for BAT1806/BIIB800 were within the equivalence margin. The 90% confidence intervals for the mean difference in sIL-6R binding by ELISA between BAT1806/BIIB800 and CN-, EU-, and US-sourced TCZ were − 4.63 to 1.78%, − 3.07 to 4.59% and − 2.92 to 3.32%, respectively, and they all fell within the equivalence acceptance criterion (− 6.50 to 6.50%, − 9.77 to 9.77%, and − 5.70 to 5.70%, respectively). Competitive inhibition of sIL-6R binding to IL-6 demonstrated similarity between BAT1806/BIIB800 and TCZ, and met the quality ranges established for this attribute. Additional characterization of the sIL-6R binding properties by SPR showed that the on-rates (ka), off-rates (kd), and equilibrium-binding constant for binding to sIL-6R are similar for BAT1806/BIIB800 and TCZ (Table 1).
Comparison of binding to sIL-6R and competitive inhibition of sIL-6R binding to IL-6 between BAT1806/BIIB800 and TCZ. a The representative dose-response curve of binding to sIL-6R. b Scatter plot of binding to sIL-6R. c The representative dose-response curve of competitive inhibition of sIL-6R binding to IL-6. d Scatter plot of competitive inhibition of sIL-6R binding to IL-6. CN China, EU European Union, IL-6R interleukin-6 receptor, Lg Log 10, sIL-6R soluble interleukin-6 receptor, STD standard (B0520180301STD, reference standard), TCZ tocilizumab, US United States
TF-1 cell-based proliferation assays (Fig. 6, Table 1) demonstrated similarity between BAT1806/BIIB800 and TCZ with statistical equivalence testing confirming that the results for BAT1806/BIIB800 were within the equivalence margin. The 90% confidence intervals for the mean difference in inhibition of TF-1 proliferation between BAT1806/BIIB800 and CN-, EU-, and US-sourced TCZ were − 12.65 to 3.00%, − 12.17 to 2.39%, and − 11.17 to 1.34%, respectively, and they all fell within the equivalence acceptance criterion (− 21.83 to 21.83%, − 19.53 to 19.53%, and − 14.20 to 14.20%, respectively). The SEAP reporter gene assay (used to assess IL-6 signalling) demonstrated that BAT1806/BIIB800 lots were within the TCZ quality range, indicating similarity between products (Fig. 6, Table 1). Across all other Fab-mediated activity bioassays, similarity was demonstrated between BAT1806/BIIB800 and TCZ as all results met the similarity criteria (Table 1).
Comparison of inhibition of IL-6-mediated proliferation in TF-1 cells and IL-6 neutralization activity (STAT3-SEAP reporter gene assay) between BAT1806/BIIB800 and TCZ. a The representative dose-response curve of inhibition of IL-6-mediated proliferation in TF-1 cells, b scatter plot of inhibition of IL-6-mediated proliferation in TF-1 cells, c the representative dose-response curve of IL-6 neutralization activity (STAT3-SEAP reporter gene assay), and d scatter plot of IL-6 neutralization activity (STAT3-SEAP reporter gene assay). CN China, EU European Union, Lg Log 10, OD optical density, RLU relative light unit, SEAP secreted embryonic alkaline phosphatase, STD standard (B0520180301STD, reference standard), TCZ tocilizumab, US United States
The Fc gamma receptor and C1q binding affinities (Fig. 7, Table 1) of BAT1806/BIIB800 and the percentage of relative binding to FcRn (Fig. 7, Table 1) of BAT1806/BIIB800 were within the quality ranges. Additionally, functional characterization of ADCC and CDC showed no activity in these assays for either BAT1806/BIIB800 or TCZ, supporting similarity.
Comparison of FcγR and C1q binding between BAT1806/BIIB800 and TCZ. Scatter plot of a binding to FcRn, b binding to C1q, c binding to FcγRIIIa (158V), and d binding to FcγRIIIa (158F). C1q complement component 1q, CN China, EU European Union, FcyRIIIa fragment crystallisable gamma receptor IIIa, FcRn neonatal fragment crystallisable receptor, TCZ tocilizumab, US Unites States
Collectively, these data demonstrate that BAT1806/BIIB800 and TCZ are similar across all Fab- and Fc-mediated analyses (Table 1).
4 Discussion
A comprehensive comparative analytical assessment was conducted with BAT1806/BIIB800 and CN-, EU-, and US-sourced TCZ using a tier system to rank and classify each quality attribute and associated methods. BAT1806/BIIB800 demonstrated similarity to TCZ in attributes directly related to the primary mechanism of action (i.e., Tier 1 attributes: binding to sIL-6R and inhibition of IL-6-mediated proliferation in TF-1 cells). Additionally, most Tier 2 (high-impact CQAs) and the majority of Tier 3 (non-critical) attributes showed BAT1806/BIIB800 to be similar to TCZ. We did observe some differences with glycosylation heterogeneity, aggregation, and charge variants. These differences were addressed during the development of BAT1806/BIIB800 through extensive structure activity relationship (SAR) studies (separate manuscript in development). Additional pharmacokinetic (PK), immunogenic, and efficacy assessments were also performed to determine any clinical impact.
Glycation is a non-enzymatic glycosylation process that may occur during manufacture, storage, and use of antibodies and could affect the structure, physicochemical properties, activity, and potential catabolism of the protein. In order to fully elucidate the differences we observed in glycation content, further SAR studies were conducted using forced glycation treatment. The studies demonstrated similar levels of modifications and trends between BAT1806/BIIB800 and TCZ that did not impact the functional bioassay activity of either molecule (data not shown). Additionally, as this modification is known to occur to proteins in vivo, no immunogenic reactions are expected and, as such, no safety concerns exist [22].
Glycan composition differed between BAT1806/BIIB800 and TCZ, with a minor increase in galactosylation, increased sialylation, decreased afucosylation, and reduced percentage of high mannose. These differences had no impact on Fc gamma receptor binding; both BAT1806/BIIB800 and TCZ did not demonstrate ADCC and CDC effects [23]. High mannose glycans have the potential to change the PK of a protein (i.e., by increasing the rate of drug clearance), therefore, having lower amounts of high mannose in the biosimilar could increase the relative half-life of the product. However, the quantitative change we observed was small (in the order of 1–2%) and no meaningful impact was anticipated, which was confirmed by non-clinical and clinical studies that demonstrated equivalent PK of BAT1806/BIIB800 in a comparative PK study [12], and similar trough serum concentrations in a phase III trial in patients with RA [13]. Therefore, differences observed in high mannose content can be considered clinically non-meaningful. Sialic acid content also has the potential to impact the PK of proteins and specifically extend the half-life of IgG though FcRn-mediated recycling [24]. We found higher levels of sialylation in BAT1806/BIIB800 compared with TCZ, but overall very low levels in both compounds, below or within the level of human endogenous IgG sialic acid content (11–15%) [25]. Based on the literature [26], the minor (< 2%) difference in levels of sialylation between BAT1806/BIIB800 and TCZ was not expected to affect the PK, bioavailability, or immunogenicity of the products [12, 13]. SAR studies confirmed that acidic fractions with up to 13% sialylation appeared to have no impact on the functional bioassay activity and FcRn binding affinity of the product (data not shown). Overall, the difference observed in sialylation can be considered clinically non-meaningful.
Particle content and aggregate profiling showed a wider range in ≥ 5-µm non-spherical particles with BAT1806/BIIB800 compared with TCZ; however, the differences reported here were within the variability of the assay. No clinically relevant differences in immunogenicity were observed in non-clinical and clinical studies [12, 13].
An increase in apparent purity was suggested as high molecular weight levels of BAT1806/BIIB800 fell below the lower limit of the quality range, whilst the monomer content was above the upper limit, but the differences were not found to be clinically meaningful and met the provisions of the EMA quality guidelines on similar biological medicinal products [7].
Analyses of charge profiles revealed that the basic region was marginally lower in BAT1806/BIIB800 compared with TCZ and the main peak was slightly higher. To better understand this difference, individual peaks were identified and SAR studies showed that the acidic and basic isoform fractions were enriched in modifications such as fragments (data not shown). BAT1806/BIIB800 and TCZ variants showed the same functional bioassay activity and FcRn binding affinities, demonstrating no impact on function (data not shown). These results indicate that the quantitative differences should have no clinical impact on the products, as no effects on the safety and efficacy of BAT1806/BIIB800 were reported [13]. Differences in hydrophobic variants were also observed between BAT1806/BIIB800 and TCZ, but SAR studies indicated these would not be clinically impactful (data not shown). Site-specific characterization of the fragments and post-translational modification variants observed were carried out in SAR studies and showed BAT1806/BIIB800 and TCZ (EU-source) HIC fractions had similar functional bioassay activity (data not shown). The combined results of the physicochemical characterization studies and functional bioassay activity analysis demonstrated that there was no major difference in these attributes between the HIC fractions, and were considered to have no impact on biosimilarity.
Overall, BAT1806/BIIB800 was found to be highly similar to TCZ in terms of structural and physicochemical properties, purity, and functional activity. Where minor differences were observed, further assessments ruled out any relevant impact on PK, efficacy, safety, or immunogenicity. In combination with the results of both non-clinical and clinical studies conducted with BAT1806/BIIB800, these data support a conclusion that BAT1806/BIIB800 is biosimilar to TCZ.
5 Conclusions
In this comprehensive comparative analytical assessment of the biosimilar BAT1806/BIIB800 and TCZ sourced from different regions, BAT1806/BIIB800 and TCZ were found to be highly similar for all critical quality attributes. Where differences were observed in less critical quality attributes, additional analytical assessments and clinical study results determined these to be not clinically meaningful.
References
Okuda Y. Review of tocilizumab in the treatment of rheumatoid arthritis. Biologics. 2008;2(1):75–82. https://doi.org/10.2147/btt.s1828.
Sebba A. Tocilizumab: the first interleukin-6-receptor inhibitor. Am J Health Syst Pharm. 2008;65(15):1413–8. https://doi.org/10.2146/ajhp070449.
American College of Rheumatology. Tocilizumab (Actemra). 2023. https://rheumatology.org/patients/tocilizumab-actemra. Accessed 18 Jan 2024.
Perrone F, Piccirillo MC, Ascierto PA, Salvarani C, Parrella R, Marata AM, et al. Tocilizumab for patients with COVID-19 pneumonia. The single-arm TOCIVID-19 prospective trial. J Transl Med. 2020;18(1):405–21. https://doi.org/10.1186/s12967-020-02573-9.
US Food and Drug Administration. Quality considerations in demonstrating biosimilarity of a therapeutic protein product to a reference product. Guidance for industry. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/quality-considerations-demonstrating-biosimilarity-therapeutic-protein-product-reference-product. Accessed 18 Jan 2024.
US Food and Drug Administration. Scientific considerations in demonstrating biosimilarity to a refence product. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/scientific-considerations-demonstrating-biosimilarity-reference-product. Accessed 18 Jan 2024.
European Medicines Agency, Committee for Medicinal Products. Guideline on similar biological medicinal products containing monoclonal antibodies—non-clinical and clinical issues. . https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-similar-biological-medicinal-products-containing-monoclonal-antibodies-non-clinical_en.pdf. Accessed 18 Jan 2024.
Center for Drug Evaluation of the National Medical Products Administration. Technical guidelines for the development and evaluation of biosimilar drugs (in Chinese). https://www.cde.org.cn/zdyz/domesticinfopage?zdyzIdCODE=f044cdf4b7d7286aa12ffb85fc81a74c. Accessed 18 Jan 2024.
World Health Organization. Guidelines on evaluation of biosimilars. Annex 3. 2022. https://cdn.who.int/media/docs/default-source/biologicals/annex-3---who-guidelines-on-evaluation-of-biosimilars---sj-ik-5-may-2022.pdf?sfvrsn=9b2fa6d2_1&download=true. Accessed 19 Feb 2024.
European Medicines Agency. Committee for Medicinal Products. Guideline on similar biological medicinal products. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-similar-biological-medicinal-products-rev1_en.pdf. Accessed 29 Apr 2024.
Berkowitz SA, Engen JR, Mazzeo JR, Jones GB. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov. 2012;11(7):527–40. https://doi.org/10.1038/nrd3746.
Zhang H, Wang H, Wei H, Chen H, Liu J, Li C, et al. A phase I clinical study comparing the tolerance, immunogenicity, and pharmacokinetics of proposed biosimilar BAT1806 and reference tocilizumab in healthy Chinese men. Front Pharmacol. 2020;11: 609522. https://doi.org/10.3389/fphar.2020.609522.
Leng X, Leszczynski P, Jeka S, Liu S, Liu H, Miakisz M, et al. Comparing tocilizumab biosimilar BAT1806/BIIB800 with reference tocilizumab in patients with moderate-to-severe rheumatoid arthritis with an inadequate response to methotrexate: a phase 3, randomised, multicentre, double-blind, active-controlled clinical trial. Lancet Rheumatol. 2024;6:E40–50. https://doi.org/10.1016/s2665-9913(23)00237-0.
US Food and Drug Administration. Development of therapeutic protein biosimilars: comparative analytical assessment and other quality-related considerations. Guidance for industry. . https://www.fda.gov/regulatory-information/search-fda-guidance-documents/development-therapeutic-protein-biosimilars-comparative-analytical-assessment-and-other-quality. Accessed 18 Jan 2024.
European Medicines Agency, Committee for Medicinal Products. Reflection paper on statistical methodology for the comparative assessment of quality attributes in drug development. . https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-statistical-methodology-comparative-assessment-quality-attributes-drug-development_en.pdf. Accessed 18 Jan 2024.
Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989;182(2):319–26. https://doi.org/10.1016/0003-2697(89)90602-7.
Rose-John S. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6. Int J Biol Sci. 2012;8(9):1237–47. https://doi.org/10.7150/ijbs.4989.
Levy DE, Lee C-K. What does STAT3 do? J Clin Investig. 2002;109(9):1143–8. https://doi.org/10.1172/JCI15650.
Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Investig. 2008;118(11):3537–45. https://doi.org/10.1172/jci36389.
Smolen JS, Beaulieu A, Rubbert-Roth A, Ramos-Remus C, Rovensky J, Alecock E, et al. Effect of interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid arthritis (OPTION study): a double-blind, placebo-controlled, randomised trial. Lancet. 2008;371(9617):987–97. https://doi.org/10.1016/s0140-6736(08)60453-5.
Dayer JM, Choy E. Therapeutic targets in rheumatoid arthritis: the interleukin-6 receptor. Rheumatology (Oxford). 2010;49(1):15–24. https://doi.org/10.1093/rheumatology/kep329.
Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology. 2011;21(7):949–59. https://doi.org/10.1093/glycob/cwr027.
Nishimoto N, Kishimoto T. Humanized antihuman IL-6 receptor antibody, tocilizumab. Handb Exp Pharmacol. 2008;181:151–60. https://doi.org/10.1007/978-3-540-73259-4_7.
Chia S, Tay SJ, Song Z, Yang Y, Walsh I, Pang KT. Enhancing pharmacokinetic and pharmacodynamic properties of recombinant therapeutic proteins by manipulation of sialic acid content. Biomed Pharmacother. 2023;163: 114757. https://doi.org/10.1016/j.biopha.2023.114757.
Boune S, Hu P, Epstein AL, Khawli LA. Principles of N-linked glycosylation variations of IgG-based therapeutics: pharmacokinetic and functional considerations. Antibodies (Basel). 2020;9(2):22. https://doi.org/10.3390/antib9020022.
Naso MF, Tam SH, Scallon BJ, Raju TS. Engineering host cell lines to reduce terminal sialylation of secreted antibodies. MAbs. 2010;2(5):519–27. https://doi.org/10.4161/mabs.2.5.13078.
Acknowledgments
Medical writing assistance was provided by Graeme Clark, PhD, Emma Beddie, BSc (Hons), and Jo Fetterman, PhD, of Parexel International and was funded by Biogen International GmbH, Baar, Switzerland.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Funding
The development of this manuscript was funded by Biogen International GmbH.
Conflict of interest
Yujie Liu, Jianhua Xie, Zhuxiang Li, Xiong Mei, Di Cao, Shengfeng Li, and Cuihua Liu are Bio-Thera Solutions Ltd employees and may hold stock, stock options, or both in Bio-Thera Solutions Ltd. Linda Engle, Suli Liu, and Hans C. Ebbers are Biogen employees and may hold stock, stock options, or both in Biogen.
Availability of data and material
Data are available upon reasonable request to the authors.
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors, and was prepared according to the International Committee of Medical Journal Editors Uniform Requirements and the International Society for Medical Publication Professionals Good Publication Practice (GPP) for Communicating Company-Sponsored Medical Research: The GPP3 Guidelines.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Code availability
Not applicable.
Author contributions
All authors contributed to the study design as well as data reporting and interpretation. All authors also reviewed and revised the manuscript. All authors participated in writing the manuscript, with the support of medical writing services provided by the funder. All authors read and approved the submitted version of the manuscript.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc/4.0/.
About this article
Cite this article
Liu, Y., Xie, J., Li, Z. et al. Demonstration of Physicochemical and Functional Similarity of the Biosimilar BAT1806/BIIB800 to Reference Tocilizumab. BioDrugs (2024). https://doi.org/10.1007/s40259-024-00662-5
Accepted:
Published:
DOI: https://doi.org/10.1007/s40259-024-00662-5