Comprehensive Physico-Chemical and Functional Similarity Assessment of Intravenous and Subcutaneous RGB-19 Drug Products as Proposed Biosimilars to Tocilizumab Reference Product

Abstract

Background and Objective

Tocilizumab is a recombinant, humanised monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which specifically targets the interleukin-6 receptor (IL-6R). The RGB-19 product was developed as a biosimilar to the reference medicinal product RoActemra^® (authorised for use in the European Union [EU]). The current study focuses on the demonstration of structural, physico-chemical and functional similarity between RGB-19 (intravenous [IV] and subcutaneous [SC] presentations) and EU-sourced RoActemra^® (IV and SC presentations).

Methods

The RGB-19 biosimilar tocilizumab product was comprehensively tested using an extensive state-of-the-art analytical and functional panel of 44 methods to demonstrate similarity to the EU-sourced RoActemra^®. Biosimilarity was rigorously evaluated through an extensive array of orthogonal physico-chemical and functional assays, supplemented by a detailed comparative characterisation of the primary and higher order structures of the therapeutic proteins.

Results

Extensive structural analyses confirmed that the primary and higher order structures of tocilizumab proteins in RGB-19 IV and SC drug products are identical or exhibit a high degree of similarity to those of the RoActemra^® reference products. The impurity profiles of RGB-19 and RoActemra^® products were found to be highly comparable, as demonstrated by a series of physico-chemical techniques. A high level of similarity was shown for the most critical (soluble IL-6R binding and cell-based anti-proliferation assay) and for all other bioassay attributes. Based on the statistical evaluation, negligible differences could be detected for sialylation, glycation, fragments and charge variants, which do not affect the functional properties.

Conclusion

Based on the similarity study, RGB-19 and RoActemra^® can be considered highly similar drug products. The minor differences found for some physico-chemical attributes do not affect the biological potency, binding and other critical attributes, and are therefore not considered clinically meaningful.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40259-025-00734-0.

Key Points

A comprehensive analytical and functional similarity study was carried out for Gedeon Richter’s biosimilar tocilizumab RGB-19 and RoActemra® reference drug products.

Based on the comparative assessment, RGB-19 and RoActemra® are highly similar drug products, except for clinically not-relevant minor differences in a few quality attributes.

Introduction

RGB-19 tocilizumab is an intended biosimilar drug product of the reference medicinal product (RMP) RoActemra^® (trade name in the European Union [EU]). Tocilizumab is a recombinant humanised monoclonal antibody of the immunoglobulin (Ig) G1 k subclass, which binds to the human interleukin-6 receptor (IL-6R) [1, 2]. Tocilizumab inhibits IL-6 signalling by binding specifically to both soluble IL-6R (sIL-6R) and membrane-bound IL-6R (mIL-6R).

The RMP was approved by the European Medicines Agency (EMA) [3] and the US Food and Drug Administration (FDA) [4] for the treatment of rheumatoid arthritis, systemic juvenile idiopathic arthritis (sJIA) and polyarticular juvenile idiopathic arthritis (pJIA), a severe form of arthritis in children. Tocilizumab was also granted authorisation for the treatment of cytokine release syndrome (CRS) in adult patients hospitalised with coronavirus disease 2019 (COVID-19).

Owing to their structural complexity, inherent (manufacturing process-derived) heterogeneity and large set of possible post-translational modifications, monoclonal antibody biosimilars are much more challenging to characterise than small molecule generic drugs [5, 6]. A high degree of similarity in clinical outcomes—both in terms of efficacy and safety—between a biosimilar candidate and its reference product presupposes a high degree of similarity in their physico-chemical and functional properties in the biosimilar regulatory framework. Considering the above, to successfully receive regulatory approval for a biosimilar product, extensive analytical and clinical comparability studies are required to prove that the biosimilar candidate is highly similar to the RMP in terms of quality, efficacy, safety and immunogenicity.

In accordance with the biosimilar development guidelines issued by the FDA [7] and EMA [8–10], it is strongly recommended to adopt a totality-of-evidence approach when designing the analytical similarity assessment programme. This comprehensive strategy is underpinned by the early identification and selection of critical quality attributes (CQAs), as outlined in the International Council for Harmonisation (ICH) guideline Q8 and other relevant regulatory frameworks [8–10]. The CQAs are determined by a risk-based methodology [11], evaluating the potential impact and associated uncertainty of each quality attribute on pharmacokinetics/pharmacodynamics (PK/PD), efficacy, immunogenicity and safety characteristics. Extensive structural, physico-chemical and biological functional testing of the reference medicinal batches (quality target product profile [QTPP] data collection covering a wide, at least 2- or 3-year time range), involving all CQAs and supporting non-CQAs, is the basis for the development of a biosimilar drug product. According to the totality-of-evidence approach, if a difference is detected in some quality attributes, additional analytical, non-clinical or clinical data can be used to demonstrate that the difference has no clinically meaningful impact on efficacy, safety or immunogenicity. This scientifically rigorous approach facilitates a well-established demonstration of biosimilarity, aligning with regulatory requirements.

The current study focuses on the analytical and functional similarity evaluation of the biosimilar RGB-19 and RoActemra^® reference drug products. The RGB-19 and the RoActemra^® drug products are available as a 20 mg/mL concentrate for solution for infusion, for intravenous (IV) use, and a 162 mg/0.9 mL (180 mg/mL) solution for injection, for subcutaneous (SC) use. These two presentations of the drug products differ in their formulation, final concentration and container closure system; thus, two separate similarity assessments are performed for the RGB-19 IV versus RoActemra^® IV and for RGB-19 SC versus RoActemra^® SC products. As the RMPs RoActemra^® IV and SC are approved for use in the EU and RGB-19 IV and SC are likewise intended to be introduced to the EU market, their development was carried out in full compliance with EMA requirements for biosimilar medicinal products.

Materials and Methods

RGB-19 product development was carried out over the course of several years using an extensive panel of state-of-the-art molecular and functional methods to demonstrate analytical similarity to the RoActemra^® reference product. All methods used in the similarity study were in their final validated or qualified state. Altogether, 44 analytical methods and functional assays (supplementary Table 1; see the electronic supplementary material) were selected based on their sensitivity and capability to detect differences in the CQAs and used during the similarity assessment. The descriptions of the major and critical methods and the obtained results are given in this publication, while the remaining descriptions and data are provided in the electronic supplementary material, as indicated in supplementary Table 1. Based on the literature of biosimilarity analytical and functional studies [12–18], a higher-than-average number of methods was applied in the current study. Each method was carefully selected following an extensive review of scientific and regulatory sources, informed by the company’s prior experience in IgG1 characterisation strategies. The method package included a large number of structural characterisation methods providing deep insights into minor molecular features and an understanding of differences experienced in physico-chemical measurements.

Data Evaluation Strategy for Similarity Assessment

For data analysis, two approaches were used to statistically evaluate the experimental results. For the quantitative results of the physico-chemical and bioassay measurements, the range approach statistical procedure was used. The quality range calculation was based on the RoActemra^®(Mean) ± X*SD formula, determined from the results of the analysis of available EU-sourced RoActemra^® batches measured continuously during the development and validation phase of the project (mean value of the RoActemra^® QTPP data). The “SD” is the standard deviation of the RoActemra^® QTPP data, and the “X” value is the multiplier, which was determined to be 3 for the majority of physico-chemical and bioassay methods and determined to be 2.5 for the most critical release bioassay methods (cell-based anti-proliferation assay and sIL-6R binding [enzyme-linked immunosorbent assay {ELISA}]), which are based on the clinically relevant mechanism of action of the product. Because of the different formulations and drug product manufacturing processes, separate quality ranges were defined for the RGB-19 SC and IV drug products. For some quality attributes such as N-glycan forms and sialic acid content, which are not dependent on the formulation and the drug product manufacturing process, a quality range combined from SC and IV RoActemra^® data was used.

The results of structural characterisation identity methods, including intact and subunit analyses, were evaluated by parallel analyses and compared to theoretical values. No multiplier was defined for negative functional assays, some biophysical and sub-visible particle methods.

Overall, 47 EU-sourced RoActemra^® IV and 33 EU-sourced RoActemra^® SC batches were analysed during the product development and validation phase. The RoActemra^® batches were measured over several years to determine the target product profile and quality ranges. In all cases, the measurements were performed within the expiry date of the RoActemra^® RMP batches. The final similarity study for marketing authorisation application (MAA) submission was performed with six RGB-19 IV and five RGB-19 SC drug product batches, which were produced from independent drug substance batches. In line with the EMA guidelines [9, 10], all batches of RGB-19 drug product that were used for the analytical similarity assessment study were manufactured using the final commercial process and scale.

Materials

The biosimilar candidate RGB-19 IV (20 mg/mL concentrate for solution for infusion) and SC (162 mg/0.9 mL solution for injection) drug products were produced by Gedeon Richter Plc. The RoActemra^® RMPs were purchased from Roche Registration GmbH and stored and handled according to the EMA summary of product characteristics document [3]. For the comparative analyses, the drug products were taken from their original containers; the active substance was not extracted prior to analysis.

Analytical Methods

Intact and Subunit Molecular Mass Analysis and Intact Glycation Analysis by Liquid Chromatography–Mass Spectrometry (LC–MS)

Intact mass analysis was performed after a dilution step on a Shimadzu Nexera X2 ultra-high-performance liquid chromatography (UHPLC) instrument coupled to a Bruker maXis II Q-TOF mass spectrometer. The on-line desalting of intact samples was performed on a Waters Acquity BEH C4 column (2.1 × 100 mm, 1.7 µm, 300 Å) at 80 ºC. For the separation, gradient elution was applied (eluent A%: H₂O = 100 [V/V%]; eluent B%: H₂O:acetonitrile [ACN] = 10:90 [V/V%]; eluent C%: ACN = 100 [V/V%]; eluent D%: H₂O:concentrated [cc.] trifluoroacetic acid [TFA] = 100:1 [V/V%]) and the flow rate was 0.4 mL/min. The same chromatographic conditions were applied for the intact glycation analysis where the samples were diluted and additionally treated with PNGaseF (New England BioLabs) and CPB (Sigma) enzymes. For the subunit analysis, the samples were digested by IdeS enzyme (Genovis) and were reduced using dithiothreitol (DTT) under denaturing conditions. The separation of the subunits (Fc/2, light chain [LC] and Fd’) was performed on the same high-performance liquid chromatography (HPLC) column at 68 ºC, with a flow rate of 0.33 mL/min, using gradient elution (eluent A%: H₂O = 100 [V/V%]; eluent B%: H₂O:ACN = 10:90 [V/V%]; eluent C%: H₂O:cc. formic acid [FA] = 100:1 [V/V%]; eluent D%: H₂O:cc. TFA = 100:1 [V/V%]). The spectra were averaged and deconvoluted using the BioPharma Compass software (Bruker). The deconvoluted mass spectra, the measured average mass (intact) or monoisotopic mass (subunit) values were compared. For glycation analysis, the relative intensity of the mono-glycated peak was determined.

Reduced and Non-reduced Peptide Mapping Analysis by LC-MS (with Lys-C and Trypsin Enzymes)

For reduced peptide mapping, the samples were first denatured at neutral pH, then digested with Lys-C enzyme (Fujifilm Wako Pure Chemical Corporation). Finally, the disulfide bridges were reduced using tris(2-carboxyethyl)phosphine (TCEP). As a first step of the non-reduced peptide mapping, the samples were denatured at neutral pH. The free thiol groups were labelled with N-ethylmaleimide (NEM). The samples were digested by Lys-C (Fujifilm Wako Pure Chemical Corporation) and Trypsin (Promega) enzymes. Finally, the digestion was stopped by the addition of acetic acid. The peptides were separated on a Waters Acquity BEH Phenyl column (1.7 μm, 2.1 × 150 mm, 130 Å) at 73 ºC using gradient elution (eluent A%: H₂O:cc. TFA = 100:0.1 [V/V%]; eluent B%: H₂O:ACN:methanol [MeOH]:cc. TFA = 20:50:30:0.1 [V/V%]). The flow rate was 0.31 mL/min. The peptide mapping analysis was performed on a Shimadzu Nexera X2 ultra-performance liquid chromatography (UPLC) instrument coupled to an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific). Data evaluation of the MS and tandem MS (MS/MS) spectra was performed using the BioPharma Finder software (Thermo Fisher Scientific). A semi-quantitative analysis of post-translational modification (PTM) levels was also carried out.

Reduced Peptide Mapping Analysis by LC-MS (with Chymotrypsin Enzyme)

In order to achieve 100% sequence coverage of the protein at the peptide level, a digestion with Chymotrypsin (Promega) enzyme was also performed. After the denaturation of the protein at neutral pH, the samples were digested with Chymotrypsin enzyme and the disulfide bridges were reduced using TCEP. The peptides were separated on a Waters Acquity UHPLC Peptide CSH C18 column (1.7 μm, 2.1 × 150 mm, 130 Å) at 55 °C column temperature using gradient elution (eluent A%: H₂O:cc. FA = 100:0.1 [V/V%]; eluent B%: H₂O:ACN:cc. FA = 50:50:0.1 [V/V%]) with a flow rate of 0.3 mL/min. A Shimadzu Nexera X2 UPLC coupled to an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific) was used for the measurements.

Free Thiol Content by Ellman’s Assay

The number of free thiol groups (sulfhydryl, -SH) per protein molecule was determined quantitatively using 5,5’-dithiobis-[2-nitrobenzoic acid] (DTNB, Ellman’s reagent). In the rapid and stoichiometric reaction, 1 mole of 2-nitro-5-thiobenzoate (TNB²⁻) is released per 1 mole of thiol. The amount of the released TNB²⁻ was quantified using an absorbance plate reader (MTP Reader Infinite Series, Tecan). Free thiol concentrations of test samples were calculated from the N-acetyl-L-cysteine standard calibration curve.

Hotspot Peptide Mapping Analysis by Reversed Phase (RP)–Ultra-High-Performance Liquid Chromatography (UHPLC) with UV Detection (with Lys-C Enzyme)

The extent of heavy chain (HC) M254 oxidation was measured with reversed-phase chromatography. HC M254 was the most sensitive amino acid residue to oxidation during the stress studies (H₂O₂, UV light). After dilution of the sample with methionine solution, DTT was added, and Lys-C enzyme (Wako) was used for the digestion. The oxidised and native HC 251–276 peptides were separated using a Waters Acquity UPLC Peptide BEH C18 reversed phase column (150 × 2.1 mm, 1.7 µm) on a Waters Acquity H-Class UPLC. The applied chromatographic parameters were as follows: column temperature 70 °C; flow rate 0.4 mL/min; gradient elution (eluent A%: H₂O:ACN:cc. TFA = 90:10:0.1 [V/V%]; eluent B%: H₂O:ACN:cc. TFA = 50:50:0.1 [V/V%]); UV detection 215 nm. The relative amount of the peptides containing the single oxidised and the non-oxidised (native) forms of the M254 were compared using area% evaluation.

N-Glycosylation Profile by Hydrophilic Interaction Liquid Chromatography (HILIC) High-Performance Liquid Chromatography (HPLC) with Fluorescence (FL) Detection

The measurement was performed in two steps. Firstly, N-glycans were enzymatically released (PNGase, GlycoPrep^® Kit, Agilent Technologies) from the denatured proteins, and derivatised using an Instant Procaine (InstantPC^TM, GlycoPrep^® Kit, Agilent Technologies) dye, followed by the removal of the excess dye. Secondly, the purified N-glycan mixture was analysed using an Acquity UPLC BEH Glycan (1.7 μm, 2.1 × 150 mm) column (Waters) on a UPLC/UHPLC (Shimadzu or Waters) with fluorescent (FLR) detection (fluorescence excitation wavelength [λ_EX] = 285 nm, fluorescence emission wavelength [λ_EM] = 345 nm). Chromatographic parameters were as follows: column temperature 45 °C; flow rate 0.5 mL/min; gradient elution (eluent A%: 50 mM ammonium formate, pH = 4.4; eluent B%: 100% ACN). Data were acquired and processed using Empower™ 3 (Waters) or LabSolutions CS 6.88 SP1 (Shimadzu) software. As a final result, the relative amounts (area%) of the different N-glycan forms were obtained.

Analysis of Bisecting GlcNAc and Gal-α-1,3-Gal Forms by HILIC-UHPLC-FL/Electrospray Ionisation(ESI) Tandem Mass Spectrometry (MS/MS) with Exoglycosidase Digestions

Samples were prepared according to the hydrophilic interaction liquid chromatography (HILIC) HPLC with fluorescence (FL) detection released glycan protocol divided into aliquots and digested with different combination of exoglycosidases. For the identification of bisecting GlcNAc forms, the results of the α(2-3,6,8,9)-Sialidase A–, β(1-4)-Galactosidase– and β-N-acetylhexosaminidase–digested aliquots were compared to the α(2-3,6,8,9)-Sialidase A– and β(1-4)-Galactosidase–digested samples. For the identification of galactose-α(1-3)-galactose linkage–containing forms, the α(2-3,6,8,9)-Sialidase A–, β(1-4)-Galactosidase– and α(1-3,4,6)-Galactosidase–digested aliquots were compared to the α(2-3,6,8,9)-Sialidase A– and β(1-4)-Galactosidase–digested samples. The samples were analysed on a Shimadzu Nexera X2 UHPLC instrument coupled to a Bruker maXis II Q-TOF mass spectrometer. The same chromatographic conditions were applied as in the HILIC-UHPLC-FL analysis. N-glycans were identified based on their measured mass, relative retention time, MS/MS spectra and the results of the exoglycosidase digestions using DataAnalysis and BioPharma Compass software (Bruker).

Sialic Acid Content by RP-HPLC-FL

N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) contents were determined in two steps. Firstly, the sialic acids (NANA and NGNA) were released from the sugar chains by acidic hydrolysis (HCl), and derivatised using a fluorescent dye (DMB, Takara Bio Inc.). Secondly, sialic acids were separated using a Phenomenex Kinetex C8 column (2.1 × 100 mm, 2.6 µm) on a UPLC/UHPLC (Shimadzu or Waters) instrument with FL detection (λ_EX = 373 nm, λ_EM = 448 nm). Chromatographic parameters were as follows: column temperature 35 °C; flow rate 0.35 mL/min; gradient (eluent A%: MeOH:H₂O = 15:85 [V/V%]; eluent B%: H₂O:cc. TFA = 100:0.1 [V/V%]). Data based on NANA and NGNA standard (Sigma) calibration curves were acquired and processed by Empower™3 (Waters) or LabSolutions CS 6.88 SP1 (Shimadzu) software.

Glycosylation Site Analysis by LC-MS Non-reduced Peptide Mapping

Samples were digested with rapid PNGase F (New England BioLabs) enzyme. Then, the samples were treated the same way as the samples during the non-reduced peptide mapping analysis. The same chromatographic conditions were applied. The glycosylation site was determined based on the analysis of the MS/MS spectra of the deglycosylated HC_E295-R303 peptide.

Non-glycosylated Heavy Chain (NgHC) Fragment by Reducing Capillary Gel Electrophoresis Sodium Dodecyl Sulphate (R-CE-SDS)

Non-glycosylated heavy chain (NgHC) was measured on a Maurice S. (ProteinSimple) CE instrument. The fragments subjected to analysis (NgHC, HC, LC) were generated by reducing the disulfide bridges of the antibody using β-mercaptoethanol together with sodium dodecyl sulfate (Maurice CE-SDS Plus 1x Sample Buffer, ProteinSimple). Compass for iCE software (ProteinSimple v 2.1.0) was used.

Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS)

The samples were prepared by a two-step dilution, first with H₂O buffer, then with D₂O buffer, and were subjected to deuteration for different time periods. After the quenching with a low pH buffer, samples were kept in an ice bath and the samples were injected into the hydrogen–deuterium exchange (HDX)-manager module of the Waters M-Class liquid chromatograph coupled to a Waters Synapt XS^® ion mobility Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). The protein was digested on-line by a Waters Enzymate^® BEH Pepsin column (30 mm × 2.1 mm, 5 µm), and the peptides were first trapped on a Waters Acquity^® UPLC BEH C18 VanGuard Pre-column (5 mm × 2.1 mm, 1.7 µm), then separated on a Waters Acquity^® UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) and finally eluted into the mass spectrometer. Chromatographic parameters were as follows: digestion column temperature 20 °C; analytical column temperature 0.1 °C; digestion eluent flow rate 100 µL/min; analytical eluent flow rate 40 µL/min; gradient elution (eluent A%: H₂O:cc. FA = 100:0.2 [V/V%]; eluent B%: ACN:cc. FA = 100:0.2 [V/V%]). The mass spectrometer was set to positive ion – HDMS^E (ion mobility) mode. The acquired HDX-MS data were analysed in two stages: first, tocilizumab-derived peptides were identified using Waters PLGS 3.0.3 and, subsequently, Waters DynamX 3.0 software was used to calculate the absolute and relative deuteration results for the identified peptides.

Two-dimensional (2D) Nuclear Magnetic Resonance (NMR) Spectroscopy

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy-based structural fingerprinting was applied for the comparative assessment of the higher order structure on the atomic level. ¹H-¹³C heteronuclear single quantum coherence (HSQC) NMR spectra of intact protein samples in uniformised near-formulation conditions were collected at 320 K on a Bruker Avance III HD 800 MHz spectrometer, equipped with a 5-mm cryogenically cooled triple-resonance Z-pulsed field gradient TCI probe (Bruker Corporation, Billerica, MA, USA). Parameters of a gradient-selected sensitivity-enhanced HSQC pulse sequence were optimised for methyl groups. 2D methyl-HSQC spectra were compared visually, as well as by a chemometric approach: sets of combined chemical shift difference (CCSD) values of pairwise comparisons of peak lists were evaluated statistically [19].

High Molecular Weight (HMW) Species by Size Exclusion Chromatography (SEC) (SEC-HPLC)

Chromatographic resolution was achieved with a Tosoh Bioscience TSKgel G3000SWXL column (7.8 × 300 mm, 5 µm, Tosoh) together with a Tosoh Bioscience TSKgel SWXL Guard precolumn (6.0 × 40 mm, 7 µm, Tosoh) on a Shimadzu Nexera HPLC system. Chromatographic parameters were as follows: column temperature 21 °C; flow rate 0.5 mL/min; isocratic flow (eluent: mix of solvent A: 100 mM NaH₂PO₄, 250 mM Na₂SO₄; solvent B: 100 mM Na₂HPO₄, 250 mM Na₂SO₄, pH 6.8); UV detection 280 nm. Data were acquired and processed by Empower™3 (Waters) or LabSolutions CS 6.88 SP1 (Shimadzu) software.

Low Molecular Weight (LMW) Species by Non-reducing Capillary Electrophoresis Sodium Dodecyl Sulphate (NR-CE-SDS)

The Maurice S. (ProteinSimple) CE instrument was used with Compass for iCE software (ProteinSimple v 2.1.0). Samples were denatured with sodium dodecyl sulfate (Maurice CE-SDS Plus 1x Sample Buffer, ProteinSimple) and acetylated with iodoacetamide. The relative amount of low molecular weight (LMW) fragments was summed as the result.

Charge Variants by Ion Exchange Chromatography (IEX-HPLC) with Native Treatment or CPB Digestion

Charge variants were separated on a strong cation-exchange column (YMC-BioPro SP-F, 100 mm × 4.6 mm, 5 µm) using a multi-step salt gradient on a Shimadzu Nexera HPLC instrument. Chromatographic parameters were as follows: column temperature 25 °C; flow rate 0.85 mL/min; gradient (eluent A: 25 mM MES buffer; eluent B: 25 mM MES buffer + 200 mM NaCl), FL detection (λ_EX = 280nm, λ_EM = 350 nm). The identity evaluation was based on the retention time values of the main peak. Using as a purity method, area% data of the main peak and sum of acidic and basic variants were evaluated.

Functional Assays

Binding to Soluble Interleukin-6 Receptor (sIL-6R) by Enzyme-Linked Immunosorbent Assay (ELISA)

Recombinant human sIL-6R was coated onto a 96-well plate. After a blocking step, serial dilutions in three replicates were prepared and transferred onto the coated plates. Binding was detected using horseradish peroxidase (HRP)-conjugated anti-human IgG (Fc-specific) followed by 3,3',5,5'-Tetramethylbenzidine (TMB) reagent. The emerging enzymatic reaction was stopped by adding sulfuric acid. Dose-response data were fit to a 4-parameter logistic (4PL) curve using PLA software (Stegmann System). The relative binding activity of the test sample was calculated from half maximal effective concentration (EC50) values derived from the dose-response curve, expressed as a relative percentage of the reference standard.

Kinetic Analysis of sIL-6R, Neonatal Fc Receptor (FcRn), Fcγ Receptors (FcγRI/CD64, FcγRIIa/CD32a, FcγRIIIa/CD16a) and the Complement Component 1q (C1q) Binding by Biolayer Interferometry (BLI)

The kinetic constants were determined by biolayer interferometry (BLI) using Octet RED96e instrument (Sartorius). As ligands, biotin-labelled human sIL-6R (Acro Biosystems), His-tagged human neonatal Fc receptor (FcRn), CD64 (Sino Biological) and CD32a 131R (R&D Systems) were immobilised on Streptavidin (SA, Sartorius) or HIS1K biosensors (Anti-Penta-HIS, Sartorius), respectively, and varying concentrations of test samples were used as analytes. In the case of FcγRIIIa/CD16a and complement component 1q (C1q) binding, test samples were immobilised on FAB2G biosensors (Anti-Human Fab-CH1 2nd Generation, Sartorius) as ligands, and varying concentrations of human CD16a 158V receptor (Acro Biosystems) and human C1q (Merck) were used as analytes, respectively. During each kinetic analysis, the sensorgrams were collected and processed using Octet Data Analysis HT software (Sartorius). The kinetic parameters of the interactions (equilibrium dissociation constant [K_D], association rate constant [k_a] and dissociation rate constant [k_dis]) were determined by fitting Langmuir 1:1 model to the kinetic curves.

Cell Surface IL-6R (Membrane-Bound IL-6R) Binding by Flow Cytometry

Human B lymphocyte-derived cell line, DS-1 (ATCC) was used, which stably and abundantly expresses IL-6R on its surface. The amount of test sample bound to IL-6R was determined by indirect immunofluorescence labelling. Samples of varying concentrations were added to the cells, and after washing steps, fluorescein isothiocyanate (FITC)-labelled anti-human IgG F(ab’)2 (Thermo Fisher Scientific) was used. The detection was performed by BD FACSVerse flow cytometer (Becton Dickinson). The results were collected by BD FACSuite software (Becton Dickinson) and evaluated using PLA software (Stegmann Systems). The measured median fluorescence intensity (MFI) values were plotted as a function of treatment concentration after log transformation. A 4PL curve was fitted to the points, and the relative biological activity of the sample was calculated from the EC50 values.

Anti-proliferation Assay

TF-1 cells (ATCC^® CRL-2003) were cultured in RPMI-1640 medium with fetal bovine serum (FBS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) before being seeded into a 96-well assay plate in the presence of FBS and recombinant human interleukin-6 (rhIL-6). Serial dilutions of the test sample were added to the IL-6–treated cells, and after incubation, viable cells were detected by adding AlamarBlue^® reagent. The relative biological activity result of the test sample was calculated from EC50 values derived from the dose-response curve with respect to reference standard, using a 4PL model fit by PLA software (Stegmann System).

Results

Most of the applied methods provided quantitative data, based on which quality ranges or at least minimum–maximum ranges could be established for the different critical and non-critical attributes to evaluate the similarity of both IV and SC presentations of the proposed biosimilar to the reference product.

Structural Characterisation

Primary Structure

Several characterisation methods were used to investigate the analytical similarity of the primary structure between the biosimilar RGB-19 and RoActemra^® reference drug products. Liquid chromatography–mass spectrometry (LC-MS)–based methods such as intact and subunit mass measurement, glycation analysis, reduced and non-reduced peptide mapping and glycosylation site analyses were performed together with free thiol determination, hotspot LC-UV peptide mapping and glycan profile analysis.

Intact mass analysis (Supplementary Fig. 1; see the electronic supplementary material) revealed the existence of the same isoforms in both presentations of the drug products. The measured average molecular mass values of the detected isoforms in RGB-19 drug products conformed to the measured molecular mass values of the RoActemra^® drug products (Supplementary Table 2). Mass analysis of the Fc/2, LC and Fd’ subunits of RGB-19 by LC-MS resulted in monoisotopic molecular masses that conform to those of the reference product (Table 1).

Table 1.

Summary of the subunit data obtained from the on-line RP-HPLC/ESI-MS analysis of the reduced and IdeS-digested tocilizumab in IV and SC drug product batches of RGB-19 and RoActemra^®. C-terminal lysine is absent, and the N-terminal glutamine amino acid of the HCs appears in pyroglutamate form

Form/sample	Theoretical monoisotopic molecular mass [Da]	Measured monoisotopic molecular mass [Da]
		RGB-19 IV	RoActemra® IV	RGB-19 SC	RoActemra® SC
Fc/2 G0F	25188.49	25188.48	25188.48	25188.49	25188.49
Fc/2 G1F	25350.54	25350.53	25350.53	25350.54	25350.54
Fc/2 G2F	25512.60	25512.58	25512.58	25512.59	25512.59
LC	23489.54	23489.52	23489.52	23489.53	23489.53
Fd’	25248.60	25248.58	25248.58	25248.59	25248.59

ESI electrospray ionisation, HC heavy chain, HPLC high-performance liquid chromatography, IV intravenous, LC light chain, MS mass spectrometry, RP reversed phase, SC subcutaneous

In order to determine the entire amino acid sequence of the protein, digestions were performed with enzymes with complementary cleavage sites, and the isobaric amino acids (Leu/Ile) were verified by multistage LC-MS/MS methods [20, 21] (methods and data not shown). Representative total ion chromatograms of the Lys-C–digested samples for each sample type are visually similar; no new or missing peaks were identified (Fig. 1a). To ensure a 100% sequence coverage of tocilizumab, digestion with chymotrypsin was also performed, resulting in similar base peak chromatogram patterns for RGB-19 and RoActemra^® drug products (Supplementary Fig. 2).

Fig. 1.

Total ion chromatograms of IV and SC drug product batches of RGB-19 and RoActemra^® obtained during the on-line RP-HPLC/ESI-MS/MS analyses of the Lys-C–digested tocilizumab under reducing conditions (a) and Lys-C+Trypsin–digested tocilizumab under non-reducing conditions (b). ESI electrospray ionisation, HPLC high-performance liquid chromatography, IV intravenous, MS/MS tandem mass spectrometry, RP reversed phase, SC subcutaneous

Altogether 16 disulfide bridges are present in the tocilizumab protein, 12 of them are intrachain disulfide bonds, while four of them connect the HCs and the LCs (Table 2).

Table 2.

Intra- and interchain disulfide bridges of tocilizumab

Type of disulfide bridge	Positions of connected cysteine residues
Intrachain disulfide bridges in the HC	Cys22-Cys96 Cys146-Cys202 Cys263-Cys323 Cys369-Cys427
Intrachain disulfide bridges in the LC	Cys23-Cys88 Cys134-Cys194
Interchain disulfide bridges between the LC and the HC	Cys222 (HC)–Cys214 (LC)
Interchain disulfide bridges between the HCs	Cys228-Cys228 Cys231-Cys231

HC heavy chain, LC light chain

The non-reducing peptide mapping method reinforced the presence of all the expected disulfide bridges (Fig. 1b). The positions of the disulfide bridges were identical between the biosimilar and the reference drug products. The free thiol content was measured by Ellman’s assay, and it was below the reporting limit of the method for both products (Table 3).

Table 3.

Summary of the quantitative analytical data related to the protein primary structure of the IV and SC presentations of the RGB-19 proposed biosimilar and reference product RoActemra^® batches

Attribute	RGB-19 IV, min–max range (n)	RoActemra® IV, quality range (n)	RGB-19 SC, min–max range (n)	RoActemra® SC, quality range (n)
Primary structure
HC M254 oxidation by RP-HPLC (rel. area%)	1.52–1.68 (6)	1.19–2.37 (46)	1.30–1.59 (5)	1.19–2.09 (28)
Oxidation in CDR by LC-MS (rel. area%)
HC_N77-K123 (M106)	1.90–2.02 (6)	1.72–1.85 (8)*	0.63–0.84 (5)	0.51–0.63 (8)*
Oxidation in non-CDR by LC-MS (rel. area%)
HC_S66-K76 (M70)	0.58–0.69 (6)	0.22–0.25 (8)*	0.17–0.27 (5)	0.08–0.10 (8)*
HC_D251-K276 (M254)	1.82–1.92 (6)	1.62–1.96 (8)*	1.24–1.49 (5)	1.31–1.83 (8)*
HC_S417-K441 (M430)	0.86–1.01 (6)	0.58–0.70 (8)*	0.62–0.73 (5)	0.46–0.55 (8)*
LC_D1-K39 (M4)	1.34–1.79 (6)	0.99–1.40 (8)*	0.47–0.70 (5)	0.35–0.57 (8)*
Deamidation in CDR by LC-MS (rel. area%)
LC_L46-K103 (Q89/Q90/N92 within CDR)	1.51–1.61 (6)	1.44–1.63 (8)*	1.85–2.63 (5)	1.93–1.97 (8)*
Deamidation in non-CDR by LC-MS (rel. area%)
HC_N77-K123 (N77/Q78/Q111)	1.15–1.26 (6)	1.14–1.18 (8)*	1.25–1.42 (5)	1.28–1.39 (8)*
HC_D150-K212 isomerA (N161/Q177/Q198/N203/N205/N210)	0.40–0.46 (6)	0.41–0.47 (8)*	0.43–0.77 (5)	0.42–0.51 (8)*
HC_D150-K212 isomerB (N161/Q177/Q198/N203/N205/N210)	0.48–0.56 (6)	0.47–0.58 (8)*	0.47–0.59 (5)	0.42–0.57 (8)*
HC_V304-K319 isomerA (N317)	0.41–0.47 (6)	0.39–0.50 (8)*	0.54–0.75 (5)	0.69–0.75 (8)*
HC_V304-K319 isomerB (N317)	0.59–0.66 (6)	0.61–0.65 (8)*	0.66–0.79 (5)	0.77–0.83 (8)*
HC_G373-K394 (N386)	2.23–2.39 (6)	2.44–2.78 (8)*	2.22–2.57 (5)	2.85–2.98 (8)*
HC_G373-K394 (N386+N391)	2.36–2.71 (6)	2.84–3.27 (8)*	1.72–2.48 (5)	2.75–2.99 (8)*
HC_S417-K441 isomerA (N436)	0.78–0.84 (6)	0.78–0.85 (8)*	0.79–0.83 (5)	0.74–0.84 (8)*
HC_S417-K441 isomerB (N436)	0.31–0.36 (6)	0.33–0.39 (8)*	0.33–0.54 (5)	0.34–0.40 (8)*
Isomerisation in non-CDR by LC-MS (rel. area%)
HC_I201-L237	2.65 (1)	2.54 (1)*	2.52 (1)	2.25 (1)*
Terminal variants by LC-MS (rel. area%)
HC_S442-G448/G448+Lys	4.53–5.06 (6)	2.81–5.47 (8)*	4.23–5.17 (5)	2.90–4.05 (8)*
HC_S442-G448/G448+Pro amidation	1.64–1.78 (6)	1.80–2.83 (8)*	1.33–1.70 (5)	0.67–5.33 (8)*
HC_Q1-K65/N-term. Gln	N/A	N/A	ND–0.03 (5)	0.57–0.85 (8)*
Free thiol by Ellman’s assay (µM)	< 9.43 (RL)	< 9.43 (RL)	< 9.43 (RL)	< 4.71 (RL)
Cys-related variants by LC-MS (rel. area%)
Trisulfide: C222 (HC) – C214 (LC)	0.80–0.87 (6)	0.26–0.53 (8)*	0.78–0.95 (5)	0.18–0.61 (8)*
Monoglycation by LC-MS (rel. int.%)	7.5–7.9 (6)	< 3.5 (LOQ)	7.5–8.6 (5)	< 3.5 (LOQ)–4.1 (8)*

CDR complementarity-determining region, HC heavy chain, HPLC high-performance liquid chromatography, IV intravenous, LC light chain, LC-MS liquid chromatography–mass spectrometry, LOQ limit of quantification, N/A not applicable, ND not detected, rel. area% relative peak area expressed in percentage, rel. int.% relative peak intensity expressed in percentage, RL reporting limit, RP reversed phase, SC subcutaneous

*Min–max range is given; quality range was not calculated because of the limited number of samples analysed with the specific method

Results of the peptide mapping experiments were also used for identifying post-translational modifications. The same N-linked glycosylation site at Asn299 was verified for both RGB-19 and RoActemra^® drug products. The relative amounts of methionine oxidation, asparagine deamidation and aspartic acid isomerisation were determined (Table 3). Both the number and the position of these modifications were the same for the biosimilar and reference products. With regard to the terminal variants, N-terminal pyroglutamination, removal of C-terminal lysine and the amidation of proline with concurrent loss of glycine amino acid were investigated. Glycation of lysine residues was characterised by the intact mass measurement of the deglycosylated protein.

Glycosylation

For the analysis of N-glycosylation, the HILIC-UHPLC-FL method was used. The glycan structures were verified by LC-electrospray ionisation (ESI)-MS measurements. Based on the results, RGB-19 and RoActemra^® drug products have similar oligosaccharide patterns except for some minor differences. The relative content of the main galactosylated, fucosylated and afucosylated glycans, the sum of sialic acid and high mannose content were determined (Fig. 2). Similar levels were obtained for the different groups of glycan species (Table 4). Sialylated glycans were present at a very low level in both the proposed biosimilar and the reference products; for the biosimilar products, values fell to the lower limit of the quality range.

Fig. 2.

N-glycan pattern of IV and SC drug product batches of RGB-19 and RoActemra^® (colour codes: RoActemra^®: green = IV, black = SC; RGB-19: blue = IV, red = SC). a High mannose glycans, b afucosylated glycans, c galactosylated glycans and d sialylated glycans. Dashed lines: upper and lower limits of combined quality ranges. IV intravenous, SC subcutaneous

Table 4.

Summary of the quantitative analytical data related to glycosylation of the IV and SC presentations of the RGB-19 proposed biosimilar and reference product RoActemra^® batches

Attribute	RGB-19 IV, min–max range (n)	RoActemra® IV, quality range (n)	RGB-19 SC, min–max range (n)	RoActemra® SC, quality range (n)
Glycosylation and sialylation
HILIC-UHPLC-FL (area%)
Galactosylated	34.5–36.4 (6)	29.5–50.3 (72)	33.5–36.3 (5)	29.5–50.3 (72)
Fucosylated	86.3–86.7 (6)	78.8–91.7 (72)	86.5–87.1 (5)	78.8–91.7 (72)
Afucosylated	3.9–4.2 (6)	2.9–4.7 (72)	4.0–4.3 (5)	2.9–4.7 (72)
High mannose	4.0–4.2 (6)	0.6–7.9 (72)	3.9–4.2 (5)	0.6–7.9 (72)
Sialylated	1.2–1.3 (6)	1.3–2.9 (72)	1.1–1.3 (5)	1.3–2.9 (72)
Sialic acid content by RP-HPLC-FL (ng/mg protein)
NANA	91.7–108.0 (6)	95.5–214.6 (61)	79.9–90.9 (6)	95.5–214.6 (61)
NGNA	< LOQ/9.6	< LOQ/9.6	< LOQ/9.6	< LOQ/9.6
LC-MS/glycopeptide (%)
High mannose forms	2.56–2.80 (6)	2.53–5.15 (8)*	2.60–3.54 (5)	2.35–3.58 (8)*
Hybrid forms	1.43–1.65 (6)	2.32–3.69 (8)*	1.34–1.74 (5)	1.28–3.46 (8)*
Galactosylated forms	39.54–41.46 (6)	40.31–50.39 (8)*	33.73–38.48 (5)	36.83–48.13 (8)*
Afucosylated forms	4.73–5.11 (6)	4.89–6.24 (8)*	5.13–5.61 (5)	4.82–5.51 (8)*
Sialylated forms	1.05–1.22 (6)	1.62–2.11 (8)*	0.76–0.85 (5)	1.00–1.82 (8)*
NgHC by R-CE-SDS (area%)	0.7–0.8 (8)	0.4–0.9 (43)	0.7–0.8 (5)	0.4–1.0 (25)
NgHC by LC-MS (area%)	1.62–1.85 (6)	1.01–1.13 (8)*	1.18–1.35 (5)	0.68–0.95 (8)*

FL with fluorescence detection, HILIC hydrophilic interaction liquid chromatography, HPLC high-performance liquid chromatography, IV intravenous, LC-MS liquid chromatography–mass spectrometry, LOQ limit of quantification, NANA N-acetylneuraminic acid, NgHC non-glycosylated heavy chain, NGNA N-glycolylneuraminic acid, R-CE-SDS reducing capillary gel electrophoresis sodium dodecyl sulphate, RP reversed phase, SC subcutaneous, UHPLC ultra-high-performance liquid chromatography

*Min–max range is given; quality range was not calculated because of the limited number of samples analysed with the specific method

Higher Order Structure

The higher order structure was characterised by various state-of-the-art orthogonal methods. Thermal stability of the protein was characterised by (micro) differential scanning calorimetry(µDSC) technique (Supplementary Fig. 3 see the electronic supplementary material). The three thermal transition temperatures corresponding to CH2, Fab and CH3 unfolding were highly similar for RGB-19 SC and RoActemra^® SC drug products, with only minor differences detected in Tm2 and total enthalpy change in certain IV samples (Supplementary Table 3).

The secondary and tertiary structures were analysed by far UV and near UV circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopic techniques. FT-IR and CD measurements both in the far UV and near UV region resulted in visually similar spectra (Supplementary Figs. 4–7). These findings indicate that the secondary and tertiary structures of RGB-19 and RoActemra^® drug products are highly similar.

Higher order structure was also investigated by HDX-MS. The technique [22, 23] can provide insight into the in-solution tertiary structure of proteins through the dependence of the hydrogen–deuterium exchange rate of protein backbone amides on their degree of exposure to a deuterated medium. The evaluation of the fractional deuterium uptake plots (or “butterfly plots”) of the examined RGB-19 and RoActemra^® drug product batches (example shown for the comparison of IV formulations on Fig. 3 for the HC and LC of tocilizumab, respectively, while SC batches are displayed in supplementary Figs. 8 and 9) and the quantitative comparison of the deuterium uptake results (data not shown) both support the conclusion that the higher order structure of the tocilizumab drug substance in the compared RGB-19 and RoActemra^® IV and SC drug product batches showed a high degree of similarity.

Fig. 3.

Fractional deuterium uptake plot (“butterfly plot”) for the comparison of the deuterium uptakes of tocilizumab a HC and b LC peptides between the examined RGB-19 IV and RoActemra^® IV product batches. Shades of blue: RGB-19 IV fractional deuterium uptake plots acquired for four different deuteration intervals; shades of green: RoActemra^® IV fractional deuterium uptake plots acquired for four different deuteration intervals (the same as for RGB-19 IV). HC heavy chain, IV intravenous, LC light chain

As an orthogonal analytical approach to HDX-MS, 2D NMR-based structural fingerprinting [24, 25] was applied for comparative assessment of the higher order structure. As a first criterion for spectral similarity, spectra were compared visually. Fig. 4 shows that the compared spectra are very similar, although seemingly missing peaks can be observed in some cases. Careful visual inspection at a lower intensity threshold revealed that every protein-related peak is present in all spectra (data not shown). NMR data were further analysed with a simple chemometric approach: statistical evaluation of CCSD values [19]. Results of chemometric assessment for RGB-19 SC batches are shown in the electronic supplementary material.

Fig. 4.

2D ¹H-¹³C HSQC NMR spectra of SC and IV drug product batches of RGB-19 and RoActemra^®. Methyl region of the spectra is shown in shifted overlay representation. A coloured rectangle shows a magnified overlaid representation of well-resolved peaks. 2D two-dimensional, HSQC heteronuclear single quantum coherence, IV intravenous, NMR nuclear magnetic resonance, SC subcutaneous

Physico-Chemical Analysis of Product-Related Variants

The product-related variant profile of the examined RGB-19 and RoActemra^® product batches was investigated by determining size (size exclusion chromatography [SEC]-HPLC, non-reducing capillary electrophoresis sodium dodecyl sulphate [NR-CE-SDS], size exclusion chromatography with multi angle laser light scattering [SEC-MALLS], analytical ultracentrifugation [AUC]) and charge heterogeneity (ion exchange [IEX]-HPLC, capillary isoelectric focusing [cIEF]), hydrophobic variants (hydrophobic interaction chromatography [HIC]) and oxidation levels (RP-UHPLC, LC-MS). The RGB-19 and RoActemra^® batches are similar in terms of all variants.

As a result of the poor separation of the fragments from the monomer peak, the SEC-HPLC method was applied only for the control of high molecular weight (HMW) species. The monomer peak and the LMW region were integrated together. The amount of ∑HMW species in RGB-19 batches is slightly lower (Table 5), which is not a safety risk, and the SEC-HPLC profiles are similar for RoActemra^® and RGB-19 batches (Fig. 5a). Using SEC-MALLS, similar monomer and HMW molecular weight (Mw) data were obtained (supplementary Table 4 see the electronic supplementary material).

Table 5.

Summary of the quantitative analytical data associated with size- and charge-related variants of the IV and SC presentations of the RGB-19 proposed biosimilar and reference product RoActemra^® batches

Attribute	RGB-19 IV, min–max range (n)	RoActemra® IV, quality range (n)	RGB-19 SC, min–max range (n)	RoActemra® SC, quality range (n)
Size-related variants
SEC-HPLC (area%)
HMW species	0.23–0.38 (6)	0.01–1.09 (47)	0.23–0.37 (5)	0.12–0.96 (31)
Monomer + LMW speciesa	99.62–99.77 (6)	98.89–100.00 (47)	99.63–99.77 (5)	99.04–99.88 (31)
NR-CE-SDS (area%)
HC-HC-LC	2.4–2.5 (6)	0.7–1.5 (46)	2.3–2.5 (5)	0.7–1.6 (25)
HC-HC	0.4–0.5 (6)	0.2–0.7 (46)	0.3–0.4 (5)	0.2–0.6 (25)
HC-LC	0.5–0.6 (6)	N/Ab	0.5–0.6 (5)	N/Ab
LC	0.4–0.4 (6)	N/Ab	0.4–0.6 (5)	N/Ab
∑LMW species	3.8–3.9 (6)	0.8–2.3 (46)	3.7–4.1 (5)	0.5–2.6 (25)
Monomer	96.1–96.2 (6)	97.7–99.2 (46)	95.4–96.3 (5)	97.4–99.5 (25)
Charge-related variants
IEX-HPLC (area%)
Acidic variants	21.5–24.3 (6)	16.4–28.8 (43)	19.2–21.8 (5)	16.4–26.1 (29)
Basic variants	9.4–9.8 (6)	7.5–16.2 (43)	9.4–11.0 (5)	8.8–20.7 (29)
Main peak	66.0–68.9 (6)	59.5–71.6 (43)	68.5–69.8 (5)	56.6–71.5 (29)
IEX-HPLC with CPB digestion (area%)
Acidic variants	22.4–25.9 (6)	16.0–29.3 (35)	20.0–23.2 (5)	16.6–27.1 (24)
Basic variants	3.4–3.8 (6)	5.2–11.9 (35)	3.5–5.1 (5)	5.3–19.1 (24)
Main peak	70.7–73.8 (6)	61.3–76.2 (35)	72.9–74.9 (5)	58.0–74.7 (24)

HC heavy chain, HMW high molecular weight, HPLC high-performance liquid chromatography, IEX ion exchange, IV intravenous, LC light chain, LMW low molecular weight, LOQ limit of quantification, N/A not applicable, NR-CE-SDS non-reducing capillary gel electrophoresis sodium dodecyl sulphate, SC subcutaneous, SEC size exclusion chromatography

^aThe monomer peak and LMW species were integrated together

^bNot detected or under LOQ

Fig. 5.

Size- and charge-related variants of IV and SC drug product batches of RGB-19 and RoActemra^®: a SEC-HPLC chromatograms, b NR-CE-SDS electropherograms, c IEX-HPLC chromatograms—native treatment and d IEX-HPLC chromatograms – after CPB digestion, with enlarged views of charge variants. HC heavy chain, HMWs high molecular weight species, HPLC high-performance liquid chromatography, IEX ion exchange, IV intravenous, LC light chain, LMWs low molecular weight species, NgHC non-glycosylated heavy chain, NR-CE-SDS non-reducing capillary gel electrophoresis sodium dodecyl sulphate, SC subcutaneous, SEC size exclusion chromatography

The level of the LMW species and the distribution of the fragments were characterised and controlled with an orthogonal NR-CE-SDS method. The ratio of ∑LMW species was calculated from the individual values of the LC, HC-LC, HC-HC and HC-HC-LC fragment peaks (Fig. 5b). RGB-19 contains a slightly higher level of ∑LMW species as compared to RoActemra^® (Table 5). The difference noted in the levels of ∑LMW species is essentially driven by elevated levels of HC-HC-LC.

Although the levels of acidic and basic charge variants of RGB-19 are similar to RoActemra^® tested by the native IEX-HPLC method, after CPB digestion, the level of the basic variants of the RGB-19 is slightly lower than in RoActemra^® batches (Table 5). It can be seen on the native IEX-HPLC chromatogram (Fig. 5c) that the basic peaks B1 at t_R ~ 8.5 min in RGB-19 samples are slightly higher, while the levels of basic peaks B2 at t_R ~ 9.2 min are similar for all products, and the B3 basic peak at t_R ~ 10.2 min is present only in RoActemra^® SC drug product. By using CPB enzyme, the removal of Lys variants results in a lower total amount of basic variants in RGB-19 as compared to RoActemra^® (Fig. 5d, Table 5). Despite of the differences in the basic variants tested by IEX-HPLC, the isoelectric point of RGB-19 measured by the cIEF method is similar to that of the RoActemra^® batches (Supplementary Fig. 11, Table 4). Using HIC, highly similar profiles were obtained for hydrophobic variants (Supplementary Fig. 12, Table 4).

Functional Biological Activity

The functional comparability was extensively investigated using state-of-the-art Fab- and Fc-mediated binding assays, as well as cell-based bioassays, which are critical and relevant for the mechanism of action of tocilizumab. In addition, supportive negative assays were also included in the study.

The binding of IL-6 to the mIL-6R activates the classical, anti-inflammatory signalling pathway, while its binding to the soluble form (sIL-6R) activates the pro-inflammatory trans-signalling pathway in cells expressing the gp130 cell surface receptor [26]. Tocilizumab binds specifically to both soluble and membrane-bound forms of IL-6R.

The primary binding event initiating the therapeutic effect of tocilizumab is the binding to sIL-6R, which has a high impact on efficacy. The sIL-6R binding of tocilizumab was measured by ELISA to compare the binding activity of the test sample to the reference sample providing data on the relative biological activity of the drug product. The ELISA data confirmed that RGB-19 and RoActemra^® batches bind to sIL-6R in a similar manner (Fig. 6). The results of the binding kinetics measured by BLI also confirmed that the binding of multiple batches of RGB-19 and RoActemra^® to sIL-6R is similar (Fig. 7). Tocilizumab can displace IL-6 from the formed sIL-6R/IL-6 complex, which was demonstrated by complex dissociation ELISA. Data confirmed similarity between RGB-19 and RoActemra^® drug products (Supplementary Fig. 13; see the electronic supplementary material). The complex of IL-6 and sIL-6R can bind to gp130 on the cell surface and trigger IL-6–dependent signalling in cells lacking expression of mIL-6R. Inhibition of sIL-6R–mediated trans-signalling results of the RGB-19 and RoActemra^® are highly similar (Supplementary Fig. 14).

Fig. 6.

Biological activity of RoActemra^® and RGB-19 IV and SC batches tested by sIL-6R ELISA. Representative dose-response curves of a RoActemra^® IV and b RGB-19 IV; c comparison of biological activity of RoActemra^® IV and RGB-19 IV batches. Representative dose-response curves of d RoActemra^® SC and e RGB-19 SC; f comparison of biological activity of RoActemra^® SC and RGB-19 SC batches. Dashed lines: upper and lower limits of quality ranges. ELISA enzyme-linked immunosorbent assay, IV intravenous, SC subcutaneous, sIL-6R soluble interleukin-6 receptor

Fig. 7.

sIL-6R interaction of RoActemra^® and RGB-19 IV and SC batches by BLI. Representative sensorgrams of a RoActemra^® IV, b RGB-19 IV, c RoActemra^® SC and d RGB-19 SC. Comparison of the kinetic parameters of sIL-6R interaction: e K_D of RoActemra^® IV and RGB-19 IV batches; f k_a of RoActemra^® IV and RGB-19 IV batches; g k_dis of RoActemra^® IV and RGB-19 IV batches; h K_D of RoActemra^® SC and RGB-19 SC batches; i k_a of RoActemra^® SC and RGB-19 SC batches; j k_dis of RoActemra^® SC and RGB-19 SC batches. Dashed lines: upper and lower limits of quality ranges. BLI biolayer interferometry, IV intravenous, SC subcutaneous, sIL-6R soluble interleukin-6 receptor

The binding of tocilizumab to mIL-6R was investigated by flow cytometry, and the dose-response curves of the batches were compared to that of the reference standard to determine relative potencies. Data confirms that the RGB-19 and RoActemra^® products show similar mIL-6R binding (Fig. 8). The activation of the signalling pathways via IL-6/mIL-6R binding was investigated by STAT3 phosphorylation. Tocilizumab inhibits the binding of IL-6 to the receptor leading to the reduction of pSTAT3 protein detected by ELISA. The inhibition results were also highly similar for both RGB-19 and RoActemra^® drug products (Supplementary Fig. 15).

Fig. 8.

Cell surface mIL-6R binding of RoActemra^® and RGB-19 IV and SC batches tested by flow cytometry-based assay. Representative dose-response curves of a RoActemra^® IV and b RGB-19 IV; c comparison of cell surface mIL-6R binding of RoActemra^® IV and RGB-19 IV batches. Representative dose-response curves of d RoActemra^® SC and e RGB-19 SC; f comparison of cell surface mIL-6R binding of RoActemra^® SC and RGB-19 SC batches. Dashed lines: upper and lower limits of quality ranges. IV intravenous, mIL-6R membrane-bound interleukin-6 receptor, SC subcutaneous

The anti-proliferative effect of tocilizumab is not only dependent on its binding to sIL-6R, but also on its affinity to mIL-6R [27], which was investigated by cell-based anti-proliferation assay measurements. The anti-proliferation assay results also confirmed that RGB-19 and RoActemra^® batches show similar potency (Fig. 9).

Fig. 9.

Biological activity of RoActemra^® and RGB-19 IV and SC batches tested by cell-based anti-proliferation assay. Representative dose-response curves of a RoActemra^® IV and b RGB-19 IV; c comparison of biological activity of RoActemra^® IV and RGB-19 IV batches. Representative dose-response curves of d RoActemra^® SC and e RGB-19 SC; f comparison of biological activity of RoActemra^® SC and RGB-19 SC batches. Dashed lines: upper and lower limits of quality ranges. IV intravenous, SC subcutaneous

The FcRn protects the antibodies from lysosomal degradation, resulting in improved in vivo stability (half-life) and efficacy [28]. Thus, FcRn binding has a direct effect on the PK properties of recombinant human monoclonal antibody therapeutics, making it an important part of in vitro similarity studies [29, 30]. Based on FcRn binding affinity measurements by BLI, the kinetic parameters of RGB-19 and RoActemra^® products are similar for both presentations (Fig. 10).

Fig. 10.

FcRn binding of RoActemra^® and RGB-19 IV and SC batches by BLI. Representative sensorgrams of a RoActemra^® IV, b RGB-19 IV, c RoActemra^® SC and d RGB-19 SC. Comparison of the kinetic parameters of FcRn binding: e K_D of RoActemra^® IV and RGB-19 IV batches; f k_a of RoActemra^® IV and RGB-19 IV batches; g k_dis of RoActemra^® IV and RGB-19 IV batches; h K_D of RoActemra^® SC and RGB-19 SC batches; i k_a of RoActemra^® SC and RGB-19 SC batches; j k_dis of RoActemra^® SC and RGB-19 SC batches. Dashed lines: upper and lower limits of quality ranges. BLI biolayer interferometry, FcRn neonatal Fc receptor, IV intravenous, SC subcutaneous

The IgG1 antibodies can activate complement efficiently [31]; thus the C1q binding affinity was also investigated by BLI. The comparability data (Supplementary Fig. 16) confirmed that the kinetic results of C1q binding of the RGB-19 and RoActemra^® are similar.

Although FcγR binding affinity can play a role in the effector functions of IgGs, their potential clinical impact is low. The FcγR does not significantly affect the PK properties of IgGs [32, 33]. The human FcγRI/CD64 recruits monomeric IgG1 with nanomolar affinity [34], which is in-line with the comparability results of RGB-19 and RoActemra^® (Supplementary Table 5). Between the two human FcγRIIa/CD32a polymorphisms at amino acid position 131 (R or H), the R131 variant having higher affinity for IgG1 [35] was used to determine the kinetic parameters of tocilizumab binding by BLI. The human FcγRIIIa/CD16a gene has a polymorphism at amino acid position 158. Instead of the lower affinity variant (F158), the kinetic properties of the higher affinity variant (V158) [36] were investigated by BLI. Based on the comparability study, the various Fc-gamma receptor binding (FcγRI, FcγRIIa, FcγRIIIa) affinity tested by BLI was within the similarity ranges (Supplementary Figs. 17–19).

Additionally, negative assays were performed. In vitro results showed that despite measurable FcγRIIIa/CD16a and C1q binding, tocilizumab has no significant antibody-dependent cell-mediated cytotoxicity (ADCC) and no complement-dependent cytotoxicity (CDC) activity (Supplementary Figs. 20 and 21) [37, 38].

In summary, all Fab-, Fc-mediated and negative bioassay data demonstrate a high level of similarity between RGB-19 and RoActemra^® batches for both IV and SC drug product presentations (Table 6, Supplementary Table 5). Together with the structural characterisation and physico-chemical results, the bioassays confirmed the structure-function relationship and its similarity between RGB-19 and RoActemra^®.

Table 6.

Summary of the quantitative functional assay data of the IV and SC presentations of the RGB-19 proposed biosimilar and reference product RoActemra^® batches

Attribute	RGB-19 IV, min–max range (n)	RoActemra® IV, quality range (n)	RGB-19 SC, min–max range (n)	RoActemra® SC, quality range (n)
Biological activity
sIL-6R binding by ELISA
Biological activity (rel. %)	89.7–102.6 (6)	88.6–111.8 (38)	84.0–95.3 (5)	79.4–118.2 (24)
sIL-6R interaction by BLI
KD (M)	7.10E-10–7.81E-10 (6)	3.35E–10–9.15E–10 (29)	4.68E–10–6.30E–10 (5)	3.30E–10–8.51E–10 (18)
ka (1/Ms)	2.35E+05–2.48E+05 (6)	1.87E+05–3.51E+05 (29)	2.53E+05–2.91E+05 (5)	1.89E+05–3.62E+05 (18)
kdis (1/s)	1.69E-04–1.91E-04 (6)	1.01E–04–2.32E–04 (29)	1.28E–04–1.52E–04 (5)	9.04E–05–2.34E–04 (18)
Cell surface mIL-6R binding by flow cytometry
Biological activity (rel. %)	101.3–112.0 (6)	88.7–117.3 (19)	87.5–103.7 (5)	79.6–108.3 (13)
Anti-proliferation in TF-1 cell
Biological activity (rel. %)	87.3–110.2 (6)	73.4–120.8 (34)	95.5–103.6 (5)	74.5–114.4 (20)
FcRn receptor binding affinity by BLI
KD (M)	2.65E-08–3.04E-08 (6)	1.92E–08–3.45E–08 (27)	2.63E–08–3.17E–08 (5)	1.76E–08–3.54E–08 (18)
ka (1/Ms)	5.91E+05–6.61E+05 (6)	4.97E+05–7.48E+05 (27)	5.93E+05–6.35E+05 (5)	4.97E+05–7.33E+05 (18)
kdis (1/s)	1.64E-02–1.84E-02 (6)	1.07E–02–2.27E–02 (27)	1.60E–02–1.88E–02 (5)	1.19E–02–2.04E–02 (18)

BLI biolayer interferometry, ELISA enzyme-linked immunosorbent assay, FcRn neonatal Fc receptor, IV intravenous, k_a association rate constant, K_D equilibrium dissociation constant, k_dis dissociation rate constant, mIL-6R membrane-bound interleukin-6 receptor, rel. % biological activity expressed as a percentage of the reference standard, SC subcutaneous, sIL-6R soluble interleukin-6 receptor

Protein Content and Particles

Protein concentration values of RGB-19 drug products fall within the quality ranges determined for RoActemra^® drug products (Supplementary Table 6 see the electronic supplementary material). Based on the results of light obscuration (LO) and resonant mass measurement (RMM) analyses (Supplementary Table 6), RGB-19 batches show a similar or slightly lower number of subvisible particles as compared to the RoActemra^® batches.

Discussion

RGB-19 drug product batches were extensively characterised with state-of-the-art analytical techniques, and their similarity to RoActemra^® reference product batches was assessed. Mass spectrometric measurements confirmed that the amino acid sequences of RGB-19 and RoActemra^® drug product batches are identical; it was verified with a sequence coverage of 100% for both the HCs and LCs. The glycosylation site was identified at Asn299. Minor differences in post-translational modifications were found, as discussed below.

The same oxidation sites were identified by LC-MS peptide mapping for both products, and the rate of oxidation for HC_Met106 (located in the CDR) was similar. For the oxidation of HC_Met70, HC_Met430 and LC_Met4, slightly higher values were measured for the proposed biosimilar than for the RMP batches. The minor differences (< 0.5 area%) detected by LC-MS methods do not pose a stability or safety risk, and do not have an impact on the biological activity, FcRn binding or aggregation (∑HMW species data), as all of these methionine sites are located in non-CDR domains. The rate of deamidation was highly similar between the biosimilar and the reference products except for the deamidation of HC_Asn386 and HC_Asn391. A slightly higher deamidation rate was observed for RoActemra^® as compared to RGB-19. However, neither of these deamidation sites is in the CDR and the differences are not considered to be clinically relevant.

LC-MS peptide mapping revealed that RGB-19 contains a slightly higher level of C-terminal lysine than RoActemra^®. As in serum C-terminal lysine is cleaved enzymatically, this difference was not expected to affect either the antigen binding or potency [39], and this assumption was supported by results of the bioassay measurements.

Glycation [40] was studied by the intact LC-MS method, and it was found that the level of monoglycated forms was higher for RGB-19 than for RoActemra^®. Although glycation can affect the stability and potency of the proteins and can increase the formation of aggregates [41], the higher level of glycation in RGB-19 had no detectable effects on Fc effector functions based on the analysis of CDC and ADCC, and did not have any impact on Fab-mediated biological activity, sIL-6R binding (ELISA and BLI), cell-based anti-proliferation assay, or any other FcRn and FcγRs binding results. In addition, it did not have an impact on the higher order structure and aggregation of RGB-19 either. Thus, the difference in the level of monoglycated forms is not considered to be significant, and no impact on efficacy, safety or immunogenicity would be anticipated.

The level of sialylated glycans was slightly lower in RGB-19 than in RoActemra^®, which was also confirmed by the RP-HPLC-FL method. The NANA content of RGB-19 was found to be slightly lower than that of RoActemra^®. Since the NANA is a common sialylation form in humans [42], and the absolute value of NANA content is very low in both RGB-19 and RoActemra^®, this minor difference is negligible and has no impact on efficacy or immunogenicity. Only the NGNA is considered immunogenic [42]. It is a non-human form of sialic acid and thus it may be able to trigger adverse immune reactions in higher quantities, but the NGNA level in RGB-19 is below the quantification limit, similar to RoActemra^®.

The same disulfide bridges were identified in both products with a minor trisulfide variant (Table 3). A high level of similarity in the higher order structure of the tocilizumab protein in the biosimilar and the reference drug product batches was confirmed using far/near UV CD, FT-IR and HDX-MS methods. Some visually observable differences were found between the 2D NMR spectra of RGB-19 and RoActemra^®, although every protein-related peak was present in both spectra. Some of the visual differences are due to inconsistent enrichment of excipient polysorbate-80 during NMR sample preparation. All visually observable differences could be explained with the slight differences in the excipient composition of the SC formulation of the biosimilar and the RMP. As far as µDSC measurements are concerned, similar thermodynamic stability was measured for the SC formulated RGB-19 and RoActemra^® drug products. However, for IV formulated RGB-19 drug product batches, a slight difference could be detected in the Tm2 values (Supplementary Table 3; see the electronic supplementary material) and the ΔH values were at the upper limit of the quality range. The slightly higher Tm2 and enthalpy results of RGB-19 IV compared to RoActemra^® IV may refer to stronger secondary binding forces in the biosimilar drug products [43]. Thus, this minor difference did not pose any risk and had no impact on other CQAs.

Regarding LMW species, the different levels of HC-HC-LC fragment had no impact on the biological activity of the products. PK and clearance of IgGs are mediated mainly by binding to FcRn receptors [44]. The FcRn binding site is located on the Fc region of the HC, which is not affected by the missing LC. The measurement data also confirm that there is no detectable difference between RGB-19 and RoActemra^® in terms of FcRn binding, sIL-6R binding and efficacy (anti-proliferative results). The higher amount of HC-HC-LC is not expected to influence immunogenicity, which is supported by the fact that the minor difference in levels of HC-HC-LC did not lead to aggregation, as confirmed by the ∑HMW species data.

In the IEX-HPLC chromatograms, differences can be seen in the basic regions. LC-MS–based structural characterisation studies of enriched charge variant fractions were performed, and the results show that RGB-19 contains a slightly higher level of C-terminal lysine than RoActemra^®, and the basic peaks B1 and B2 contain lysine variants. A higher level of proline amidation was determined for RoActemra^® than for RGB-19. Amidated forms are enriched in the basic peak B2. In addition, N-terminal glutamine was identified only in RoActemra^® SC in basic peak B3. Spontaneous cyclisation of N-terminal Gln results in the formation of pyroglutamate (pyroE) [39]. The presence of pyroE has no effect on antibody structure and antigen binding, and the conversion to pyroE of recombinant monoclonal antibodies continues in vivo, and pyroE naturally exists in endogenous human IgG1 [45]. The above minor differences resulted in a slightly different basic charge variant profile between RGB-19 and RoActemra^® batches, but the lack of an N-terminal glutamine variant and lower level of proline amidation in RGB-19 compared to RoActemra^® do not affect the efficacy and biological activity [39]; thus, these differences are not considered to be critical.

Conclusions

A comprehensive physico-chemical and functional similarity study was conducted for the in-depth comparison between the IV and SC drug product presentations of biosimilar candidate RGB-19 tocilizumab and EU-sourced RoActemra^® reference products. Multiple state-of-the-art methods were applied, including high-resolution structural characterisation techniques that provided explanations for the minor differences observed in physico-chemical measurements. Based on the statistical evaluation, negligible differences were detected for sialylation, fragments, glycation and charge variants of the biosimilar and reference drug products, which did not affect the higher order structure and the functional properties; thus, these differences are not considered clinically relevant. It should be noted that only minor differences were observed between the IV and SC presentations of the biosimilar candidate. The RGB-19 and RoActemra^® demonstrated a high level of similarity for all the bioassay attributes examined in this study. Based on the evaluation of the above physico-chemical and functional similarity results, RGB-19 and RoActemra^® can be considered highly similar tocilizumab drug products and thus are expected to have comparable efficacy and safety profiles. This expectation has been proven by successfully completed phase I and phase III clinical trials (publication of results in progress).

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Employment

All authors are employed by Gedeon Richter Plc.

Conflict of Interest

The authors acknowledge potential conflicts of interest as current employees of Gedeon Richter Plc.

Funding

This study was funded by Gedeon Richter Plc.

Availability of Data and Material

The authors confirm that the relevant data supporting the findings of this study are available within the article and its supplementary material.

Ethics Approval

No studies with human participants or animals were carried out during this study. The suppliers of the human cell lines used in this study are indicated in the relevant sections of the article.

Consent to Participate/Publish

Not applicable.

Code availability

Not applicable.

Authors’ Contributions

VH and KS developed the concept of this study. ST, TF, AI and RH designed and performed experiments. All authors participated in the collection, evaluation and discussion of the results and contributed text and figures to the manuscript. All authors read the manuscript and approved its publication.