Adalimumab-aqvh/CHS-1420 (YUSIMRYTM) (hereafter referred to as adalimumab-aqvh) was recently approved by the US Food and Drug Administration as a biosimilar for adalimumab.
Objective
The current study was conducted to investigate the analytical similarity of adalimumab-aqvh and the reference product, adalimumab.
Methods
The structural, functional, and stability attributes of adalimumab-aqvh and adalimumab were compared using state-of-the-art assays. The primary structure, disulfide structure, glycan profile, secondary and tertiary structures, molar mass, size variants, free thiol, charge variants, hydrophobic variants, post-translational modifications, subvisible particles, host cell proteins, and protein concentration were investigated. The functional similarity between adalimumab-aqvh and adalimumab was demonstrated by comparing fragment antigen-binding (Fab)-associated and fragment crystallizable (Fc)-associated biological activities. The stability of adalimumab-aqvh and of adalimumab was compared through forced degradation.
Results
The structural attributes of adalimumab-aqvh were identical to those of adalimumab or met the similarity criteria, with a few exceptions. Adalimumab-aqvh and adalimumab exhibited comparable stability profiles and functional activities. Any observed differences in the physiochemical attributes did not impact the conclusion of similarity because they did not influence any functional activities related to the adalimumab mechanism of action.
Conclusion
The structural, functional, and stability data provide convincing evidence of biosimilarity between adalimumab-aqvh and the reference product, adalimumab.
Yijia Jiang and Taruna Arora are co-first authors.
Key Points
Biosimilars require extensive testing to demonstrate that structural and functional attributes are comparable to those of the reference products.
Adalimumab-aqvh was recently approved as a biosimilar to adalimumab based in part on the extensive comparative analysis of these attributes.
Minor differences observed in the structural and functional comparison did not impact the overall conclusion of similarity between adalimumab-aqvh and adalimumab.
1 Introduction
Adalimumab-aqvh (YUSIMRY™, Coherus BioSciences) was recently approved by the US Food and Drug Administration (FDA) as a biosimilar for adalimumab (HUMIRA®, AbbVie) [1]. Adalimumab-aqvh is approved for most of the same indications as adalimumab: rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, Crohn’s disease, and hidradenitis suppurativa [2, 3]. Similar to adalimumab, adalimumab-aqvh is an immunoglobulin G1 recombinant monoclonal antibody; its primary mechanism of action is to bind tumor necrosis factor α (TNF-α) and neutralize its activity by blocking the binding to TNF receptors I and II (TNFRI and TNFRII) [4]. Additionally, adalimumab and adalimumab-aqvh mediate the induction of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by binding to fragment crystallizable (Fc) gamma receptors and complement protein C1q. This mechanism of action may be associated with the inflammatory bowel disease-related action of adalimumab [5].
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Biosimilars are biologic drugs that, after thorough evaluation through an established process set up by the FDA, are highly similar to and deemed to have no clinically meaningful differences from the reference product [6]. Biosimilars are generally provided at a lower cost than the reference product, potentially increasing treatment access for patients and encouraging market competition [7].
The determination of biosimilarity between a reference product and a biosimilar requires a totality of evidence approach in a stepwise fashion. The first step and a key aspect of this evidence is the comparative analytical assessment of the physicochemical and functional attributes—including quality attributes based on the mechanism of action—between the reference product and the biosimilar [8]. Although adalimumab-aqvh and adalimumab have the same primary structure and sequence, some protein heterogeneity could be expected because of the inherent variation that can occur when biologic products are manufactured. Minor differences as a result of post-translational modifications might occur during the production of biologics in living cells [6].
The current study presents the extensive assessments performed to ensure that adalimumab-aqvh demonstrates analytical (physiochemical and functional) similarity to the reference product adalimumab. The results of the current study were part of the Biologics License Application that, in combination with clinical safety and efficacy studies [9] led to the approval of adalimumab-aqvh as a biosimilar to adalimumab [1].
2 Materials and Methods
2.1 Materials
Structural and functional assessments were performed on up to 17 independent lots of adalimumab-aqvh and up to 43 lots of commercially available US adalimumab with expiry dates spanning more than 6 years.
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2.2 Methods
Statistical comparisons of adalimumab-aqvh with the reference product were made for quantitative results to establish whether acceptance criteria were met. Statistical analyses were carried out using two approaches based on risk ranking of the product quality attributes. The most critical attributes with highest risk to clinical outcome were compared using equivalence tests based on standard deviation and confidence intervals derived from adalimumab lots [10, 11]. Equivalence is shown if the 90% two-sided confidence interval of the difference between means for adalimumab-aqvh and adalimumab is within the equivalence acceptance criterion of ± 1.5 σ based on the standard deviation (σ) calculated from tested adalimumab lots. The remaining quantitative quality attributes were compared using a quality range. The quality range is defined as the mean ± k × standard deviation of adalimumab results, with k = 3. Similarity is shown if at least 90% of adalimumab-aqvh results are within the one-sided (mean ± 3 standard deviation) or two-sided (mean ± 3 standard deviations) limits, as appropriate. The value of k is chosen as 3 consistent with a normal distribution where 99.73% of the population is included within three standard deviations of the population mean.
The methods used for the comparative analytical assessment are described in the following sections. These methods were qualified and shown to be fit for purpose (data not shown). The adalimumab-aqvh primary reference standard lot was used as the reference standard for all functional testing for which relative activity was used to assess similarity. Adalimumab-aqvh samples were analyzed together with reference product samples wherever possible.
2.2.1 Reduced Peptide Mapping
Reduced peptide mapping with trypsin digestion and liquid chromatography with tandem mass spectrometry (LC–MS/MS ) was performed on a ThermoFisher Scientific Orbitrap Q Exactive coupled to an ultra performance liquid chromatography (UPLC) system. Peptides were separated on a ThermoFisher Scientific Acclaim Vanquish C18 column (2.2 µm, 2.1 mm × 250 mm) at 25 °C using a gradient of acetonitrile/water containing formic acid, 0.1%, at a flow rate of 0.3 mL with UV absorbance detection at 215 nm. MS was performed in positive mode at an Orbitrap resolution of 60,000 over a range of 200–2000 mass to charge ratio (m/z). MS/MS was achieved by 28% higher-energy collisional dissociation energy and detected by Orbitrap by auto-scan range. Data were processed using ThermoFisher Scientific BioPharma Finder software, version 3.2, to identify peptide fragments.
2.2.2 Intact Mass
Intact, reduced, and deglycosylated protein samples were injected onto a Waters Mass PREP Micro desalting column (2.1 × 5 mm, 20 μm particle). A gradient of acetonitrile/water containing formic acid, 0.1%, was used. Mass spectra were acquired in positive ion polarity mode using an electrospray ion source kept at 125 °C (desolvation temperature of 450 °C); capillary voltage was set at 2.5 kV.
2.2.3 Non-Reduced Peptide Mapping
For the non-reduced peptide mapping, samples were treated with trypsin under non-reducing conditions. LC–MS/MS was performed using a ThermoFisher Scientific Orbitrap Fusion Lumos Tribrid high-resolution mass spectrometer coupled to a UPLC system. The disulfide-linked peptides were separated using a ThermoFisher Scientific Acclaim Vanquish C18 UPLC reversed-phase column (2.1 × 250 mm, 2.2 μm particle) and a gradient of acetonitrile/water containing formic acid, 0.1%, at a flow rate of 0.3 mL with UV absorbance detection at 215 nm. MS was performed from m/z 197 to 2000 Da under the positive polarity mode by Orbitrap resolution of 60,000. MS/MS was performed by 28% higher-energy collisional dissociation energy and detected by Ion Trap from m/z 120. Data were processed using ThermoFisher Scientific BioPharma Finder software, version 3.2, to identify peptide fragments.
2.2.4 FTIR Spectroscopy
An FTIR spectrometer equipped with a room temperature deuterated triglycine sulfate detector/liquid nitrogen cooled mercury cadmium telluride detector was used. Protein samples were concentrated to ~ 90 mg/mL and placed between two CaF2 windows with a 6- to 7-µm spacer. Spectra were collected at 4 cm−1 resolution, with a data average of 256 scans. A buffer (filtrate from concentration of protein samples) spectrum and residual moisture peaks were subtracted. Second-derivative spectra were calculated using the Savitzky–Golay method, with a second order of polynomial function.
2.2.5 Far and Near UV CD
Samples were diluted to 1 mg/mL in a common formulation buffer for far and near UV circular dichroism (CD) analysis using a CD spectrophotometer. Measurements were carried out at room temperature using 1 cm/cell and 0.02 cm/cell for near and far UV CD, respectively. After subtracting the buffer spectrum, the CD spectrum of each sample was converted to the mean residue ellipticity using the mean residue molecular weight of 109.35 and the path length of the cell.
2.2.6 Intrinsic Fluorescence
Each sample was diluted to 0.1–0.3 mg/mL in a common formulation buffer. Excitation was at 280 nm, and intrinsic fluorescence data was collected from 280 to 450 nm using a fluorescence spectrophotometer. The excitation and emission slits were both at 5 nm, and the scan rate was 300 nm/min.
2.2.7 Differential Scanning Calorimetry
Differential scanning calorimetry analysis was conducted using a Malvern VP-Capillary DSC. The samples were diluted to 0.5 mg/mL in a common formulation buffer, loaded, and scanned from 10–95 °C at 1 °C/min with a 10-second data averaging period or 10–110 °C at 1 °C/min with an 8 second data averaging period with the formulation buffer in the reference cell. Data analysis was done using Origin software, version 7.0 (OriginLab). The heat capacity profiles were normalized to protein concentration.
2.2.8 Glycan Analysis
The protein was first bound to a protein A cartridge, washed, and digested with N-glycanase. The released glycans were centrifuged and labeled with 2-aminobenzamide (InstantAB; Prozyme). The labeled N-glycans were analyzed by hydrophilic interaction chromatography-high performance liquid Chromatography (HPLC) on a Waters X-bridge Amide 3.5 µm 2.1 × 150 mm column at 45 °C and a flow rate of 0.5 mL/min with fluorescence detection. A trifluoroacetic acid/ acetonitrile gradient was used in the mobile phase.
2.2.9 Size-Exclusion Chromatography with Multi-Angle Light Scattering
Size-exclusion chromatography with multi-angle light scattering (SEC–MALS) analysis was done using an HPLC system with a multi-angle light scattering and a refractive index detector and a Sepax SRT SEC-300 7.8 × 300 mm, 5-µm particle column at 25 °C and a flow rate of 0.7 mL/min. The running buffer was sodium phosphate 50 mM, sodium chloride 250 mM (pH 6.8). The protein load was 100–200 µg, and the molecular weight of the species was determined using a refractive index increment value of 0.185 mL/g.
Ten micrograms of sample were injected using a UPLC system and a Waters Acquity BEH-200 SEC, 1.7 µm, 4.6 × 300 mm column running at a flow rate of 0.1 mL/min and a column temperature of 25 °C. The mobile phase was sodium phosphate 100 mM, sodium sulfate 150 mM, and 1-propanol, 5% (pH 6.3).
2.2.11 Reduced and Non-Reduced Capillary Electrophoresis in Sodium Dodecyl Sulfate
Samples were analyzed using a Beckman PA800 Plus CE instrument with UV absorbance detection. Samples at 2 mg/mL were denatured under heat with sodium dodecyl sulfate (SDS). Reducing conditions were achieved by including β-mercaptoethanol, ~ 5%. Under non-reducing conditions, ~ 12 mM iodoacetamide was included to block disulfide interchange. The denatured proteins were separated electrophoretically through a bare fused-silica capillary with a 20 cm effective length and a photodiode array detector set to 220 nm.
Sedimentation velocity analytical ultracentrifugation analysis was carried out at 20 °C, 40,000 rpm for 6 h or 35,000 rpm for 7.3 h monitored at 280 nm using an analytical ultracentrifuge. The samples were diluted to 0.5 mg/mL in a common formulation buffer and loaded into cells with two-channel charcoal-epon centerpieces with a 12-mm optical path length. The dilution buffer was run in the reference channel. Data analysis was done using the NIH SEDFIT program (version 11.3/version 16.2b) to derive the distribution of sedimentation coefficient. The fluorescence intensity ratios and meniscus position were fitted to find the best overall fit of the data for each sample. A maximum entropy regularization probability of 0.683 (1σ) was used, and time-independent noise was removed. The size distribution peaks were integrated using the OriginLab Origin program, version 9.0.0.
2.2.13 Cation-Exchange Chromatography
Cation-exchange chromatography was carried out on samples before and/or after carboxypeptidase B (CPB) treatment to eliminate the influence of variable processing of the C-terminal lysine and to allow for better comparison of stability-indicating charge variants. Proteins were separated using an HPLC system and a SCX-NP5 column (4.6 × 250 mm, 5 µm), a pH gradient, and a column temperature of 30 °C at a flow rate of 0.8 mL/min. At 5 mg/mL, 20 µL of each sample was injected and detected at 280 nm. Resolved peaks were integrated and reported as acidic, main, and basic peak groups.
2.2.14 Imaged Capillary Isoelectric Focusing
Samples were prepared at 0.3 mg/mL in urea 3.6 M; methylcellulose, 0.35%; and Pharmalyte 8-10.5, 4% (Millipore Sigma), and spiked with bracketing isoelectric point (pI) markers. The pI of the main peak was determined by linear regression between the two included pI markers.
Separation was achieved using a 2.7 µm Halo column (2.1 × 150 mm); a UPLC system; a trifluoroacetic acid, water, and acetonitrile gradient; and a flow rate of 0.25 mL/min at 75 °C. Absorbance was detected at 215 nm. Samples were treated with IdeS protease, which specifically targets IgG to generate F(ab)2 and Fc fragments, and with CPB to remove heavy chain C-terminal lysine.
2.2.16 Post Translational Modifications
For quantification of post-translational modifications, reduced peptide mapping data were processed using ThermoFisher Scientific BioPharmaFinder software, version 3.2, for which unmodified and modified peptides were identified using MS/MS. The MS peak area of each m/z for the peptide containing the attribute of interest was calculated by use of the software. The percentage of modification was obtained by dividing the peak area of the peptide with the modification over the total peak area of both the modified and the unmodified peptides.
2.2.17 Free Thiol
Levels of free thiol were determined using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman’s reagent). Samples were mixed with a guanidine hydrochloride solution containing ethylenediaminetetraacetic acid 2 mM and DTNB 0.1 mM (pH 8.0). Released 2-nitro-5-thiobenzoic acid, which is proportional to the free thiols in the protein, was quantified by absorbance at 412 nm using a UV-vis spectrophotometer.
2.2.18 Host-Cell Protein
Host-cell protein content was analyzed using a commercial enzyme-linked immunosorbent assays (ELISA) kit according to the manufacturer’s instructions (CHO protein ELISA 3G kit #F550, Cygnus Technologies, Inc.).
2.2.19 Protein Concentration
Protein concentrations were determined using absorbance at 280 nm and measured using a SoloVPE variable pathlength spectrophotometer. A minimum of 20 µL of sample was loaded directly without dilution and analyzed using an experimentally determined extinction coefficient of 1.45 mg/mL·cm.
2.2.20 Subvisible Particle Analysis
Microflow imaging was carried out using a ProteinSimple MFI 5200 instrument. Drug product samples were expelled from syringes into 5-mL clean glass vials and degassed under light vacuum before analysis. A morphologic filter was applied to report particles with an aspect ratio < 0.85 to reduce interference from silicone oil droplets.
A qualified sTNF-α–indirect ELISA was used to compare binding of adalimumab-aqvh and adalimumab. Plates were coated with TNF-α, followed by addition of a dilution series of adalimumab. Anti-human IgG conjugated to horseradish peroxidase was then added to detect bound adalimumab. Plates were washed, followed by the addition of substrate (tetramethylbenzidine) to the plates. Stop solution (sulfuric acid 1 M) was added, followed by data collection. Curve fit parameters were analyzed using SoftMax Pro (Molecular Devices). Relative binding was calculated against the primary reference standard adalimumab-aqvh. The adalimumab sTNF-α ELISA design enabled evaluation of the mean relative binding result from three independently prepared assay plates for each test material.
The binding affinity was characterized by surface plasmon resonance (SPR) using a Biacore T200 SPR instrument. A biosensor chip pre-immobilized with protein A was used to capture the sample via the Fc portion of the molecules on flow cells 2, 3, and 4, with flow cell 1 used as the reference. The running buffer was 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Polysorbate 20 (HBS-EP+). Soluble TNF-α at 0.30–9.53 nM, along with buffer blanks, were injected over the surface of the protein A-bound adalimumab. The sensorgrams were fit to a 1:1 kinetic interaction model using the Biacore T200 evaluation software (version 3.2). The affinity was calculated by taking the ratio of kd/ka.
2.2.23 Apoptosis Assay
Soluble TNF-α neutralization potency was determined using a validated cell-based assay that is used to measure the ability of adalimumab to bind to TNF-α and inhibit sTNF-α–induced apoptosis in the TNF-α-sensitive human monocytic cell line. Apoptosis was detected and quantified by measuring caspase activation using a Caspase-Glo 3/7 luciferase kit (Promega), and the relative potency was measured against the adalimumab-aqvh primary reference standard.
Cells overexpressing mTNF-α were seeded on plates. To analyze samples, mTNF-α target cells were co-incubated with peripheral blood mononuclear cell (PBMC) effector cells at a ratio of 1:50. Samples were added at various dilutions and incubated for 18–22 h. Cell cytotoxicity was measured by adding CytoTox-Glo reagent (Promega) 30 min before reading the luminescence. Samples were tested in triplicate on three independently prepared assay plates. The ADCC dose-response data were imported into PLA 2.0 software and were analyzed for parallelism, regression, and linearity of the test material response compared with the reference standard. The mean relative potency [half-maximal effective concentration (EC50)] obtained from the dose-response curve and mean relative cell death were calculated.
2.2.25 Complement-Dependent Cytotoxicity Activity
The reduction in viability was measured using mTNF-α-overexpressing cells to determine CDC activities in the presence of rabbit complement. The reduction in cell viability was detected by CellTiter-Glo (Promega) as an assay readout. Samples were tested in triplicate on two independently prepared assay plates. The relative potency was analyzed using PLA 2.0 software by comparing the EC50 from the dose-response curve of the reference standard and test samples using a restricted fit and four-parameter logistic curve model.
A qualified meso-scale discovery (MSD) electrochemiluminescence assay was used to compare the binding of adalimumab-aqvh and adalimumab in CHO cells engineered to express TNF-α on the cell surface. Cells were plated, followed by blocking before the addition of reference standard and test materials. After 1 hour of incubation, MSD-conjugated detection reagent and then read buffer were added. Plates were read on the MSD Sector 2400 reader (Meso Scale Diagnostics). Data analysis was performed using SoftMax Pro and PLA 2.0 (Stegmann Systems GmbH) software. Relative mTNF-α binding was calculated against the adalimumab-aqvh primary reference standard.
2.2.27 Mixed Lymphocyte Reaction Assay
Mixed lymphocyte reaction assays were performed using PBMCs from patients with Crohn’s disease as either stimulator or responder cells with a subset of adalimumab and adalimumab-aqvh lots. PBMCs from responders were diluted to 2 × 106 cells/mL and added at 50 μL/well. The stimulators from PBMCs from healthy individuals or patients with Crohn’s disease or the monocytes were irradiated at 3000 Rad, diluted in medium to 2 × 106 cells/mL or 1 × 106 cells/mL (depending on the responder:stimulator ratio of 1:2 or 1:1), and added at 50 μL/well. After incubation for 1 h, diluted test samples were added at 50 μL/well (40 μg/mL) and incubated for 6 days. Experiments were performed in 96-well round-bottom plates in quadruplicate. Mean values for fluorescence were used to derive the percentage of inhibition of proliferation activity. For flow cytometric analysis of mTNF-α expression and macrophage phenotype, MLR reactions were set up as just described but scaled up to 12-well plates with 8 × 105 responders/well and 8 × 105 stimulators/well. After incubation for 7 days, cells were transferred into 96-well round-bottom plates for staining.
2.2.28 C1q Binding by Enzyme-Linked Immunosorbent Assay
A validated C1q direct ELISA was used to evaluate the relative binding of adalimumab-aqvh and adalimumab. Plates were coated with adalimumab dilution series and controls. After incubation, washing, and blocking, C1q was added to plates for incubation at room temperature. Plates were washed and substrate (tetramethylbenzidine) was added to the plates. Stop solution (sulfuric acid 1 M) was added and data were collected. Curve fit parameters were analyzed using SoftMax Pro. Relative binding was calculated against the primary reference standard.
2.2.29 Surface Plasmon Resonance Binding Affinity Assays for Fcγ Receptors
The affinities of Fcγ receptors (FcγRs) were measured on a Biacore 8K instrument with HBS-EP+ running buffer at pH 7.4. The method consisted of amine coupling of sTNF-α as a capture reagent for adalimumab-aqvh on all flow cells of all channels, with one flow cell being used as a reference. After capture of the reference standard or adalimumab-aqvh on flow cell 2, FcγRIIIa, FcγRIIIb, FcγRIIa, FcγRIIbc, and FcγRI were injected at 15 °C over all channels. Binding affinities were determined using either a steady state model or a fit to a 1:1 kinetic interaction model (FcγRI only).
2.2.30 Surface Plasmon Resonance Binding Affinity Assay for Neonatal Fc Receptor
The binding of FcRn to adalimumab-aqvh was determined using a Biacore T200 instrument with a running buffer of sodium phosphate 10 mM; sodium chloride 150 mM; Tween 20, 0.05%; and glycerol, 20% (pH 5.8). The method consisted of amine coupling of neutravidin in sodium acetate buffer 10 mM (pH 5.0) as a capture reagent for biotinylated FcRn on flow cells 2, 3, and 4, with flow cell 1 being the reference flow cell. After capture of biotinylated FcRn, adalimumab or reference standard was injected at 15 °C over all flow cells, and binding affinities (KD) were determined using a 1:1 kinetic interaction model. The FcRn surface was regenerated using a sodium phosphate buffer (pH 7.4).
2.2.31 Forced Degradation Under Heat Stress
Adalimumab-aqvh and adalimumab samples were subjected to dialysis into adalimumab formulation buffer to assess degradation independent of formulation differences and to equalize any dialysis effects. Samples were heated at 40 °C for up to 3 months. Several stability-indicating analytical methods [e.g., SE-UPLC and cation-exchange chromatography (CEX), same as described previously] were used to provide a comprehensive assessment of potential degradation pathways.
3 Results
State-of-the art physiochemical and functional assays were used to evaluate all relevant quality attributes of adalimumab-aqvh and the reference product (adalimumab), considering the physicochemical characteristics and the known, likely, and plausible mechanism of action of the reference product. Structural and functional assays were developed to identify and characterize any differences in product attributes and were qualified for their intended use. Forced-degradation studies were used to establish degradation profiles and to provide a direct stability comparison. The comparative analytical assessment results are presented in Table 1 (additional results that support the overall comparative assessment are summarized in Supplementary Table S1).
Table 1
Comparative structural and functional analytical assessment results.
Quality attribute
Method
Lots, adalimumab: adalimumab-aqvh
Adalimumab-aqvh range
Adalimumab range
Acceptance criteriona
Result
Primary structure
Amino acid sequence
Reduced tryptic map with LC–MS/MS
9:9
Adalimumab-aqvh map visually similar to adalimumab map
Met
Adalimumab-aqvh sequence matched adalimumab with 100% coverage
Met
Intact IgG mass
8:9
Adalimumab-aqvh matched adalimumab within experimental variability
Met
Disulfide structure
Non-reduced tryptic map with LC–MS/MS
9:9
Adalimumab-aqvh visually similar to adalimumab, with expected disulfide-linked peptides identified
Met
Higher-order structure
Secondary structure
FTIR
9:9
Second-derivative FTIR spectra similar between adalimumab-aqvh and adalimumab by visual comparison
Met
Secondary and tertiary structures
Far and near UV CD
9:9
CD spectra similar between adalimumab-aqvh and adalimumab by visual comparison
Met
Tertiary structure
Intrinsic fluorescence
9:9
Fluorescence spectra similar between adalimumab-aqvh and adalimumab by visual comparison. Maximum-intensity wavelength similar
Met
Secondary and tertiary structures
DSC
9:9
Similar melting curves between adalimumab-aqvh and adalimumab by visual comparison; similar Tm
Met
Glycan profile
High mannose, %
HILIC
42:17
8.7–16.9
9.6–14.4
7.9–15.1
Met
(1 lot out)
Total afucose, %
9.5–17.4
10.8–15.6
8.9–16.6
Met
(1 lot out)
Afucose, %
0.5–1.3
0.8–1.4
0.5–1.4
Met
tGal, %
4.7–23.3
16.3–19.5
15.3–19.9
Not met
(7 lots out)b
Sialic acid, %
0.1–0.9
0.4–1.1
0.2–1.0
Not met (4 lots out)c
Molar mass and self-association
Molecular weight of protein monomer (intact IgG) and HMW
SEC-UV/RI/MALS, kDa
9:9
Adalimumab-aqvh and adalimumab molecular weight monomer within 5% of theoretical
HMWS of adalimumab-aqvh similar to adalimumab
Met
Purity
Size variant, % HMWS
Size exclusion–UPLC
30:16
0.1–0.4
0.3–0.4
≤ 0.4%
Met
Size variant, % main peak
99.2–99.8
98.9–99.4
≥ 98.7
Size variant, % LMWS
0.1–0.4
0.2–0.8
≤ 1.0
Size variant, % main peak (intact IgG)
nrCE–SDS
30:16
94.4–97.7
96.6–97.9
≥ 96.3
Not met (9 lots out)d
Size variant, % LC+HC
Reduced CE–SDS
30:16
98.5–99.1
97.8–98.4
≥ 97.7
Met
Size variant, % NGHC
0.4–0.7
1.1–1.3
≤ 1.5
HMWS, %
AUC
9:9
% HMWS in adalimumab-aqvh similar to % HMWS adalimumab within method capability
Met
Charge variant, % acidic peak
CEX
36:16
14.3–21.8
15.8–20.2
≤ 21.2
Met (1 lot out)
Charge variant, % main peak
72.4–78.0
56.1–64.1
≥ 53.1
Met
Charge variant, % basic peak
5.3–8.6
18.3–26.3
≤ 29.1
Met
Charge variant, % acidic peak
CEX (with CPB treatment)
19:16
15.5–22.6
20.0–22.5
≤ 22.9
Met
Charge variant, % main peak
73.4–80.8
71.4–75.2
≥ 70.3
Met
Charge variant, % basic peak
3.5–4.2
4.8–7.0
≤ 7.4%
Met
Oxidation, %Fc oxidation
RPC after IdeS and CPB digestion
16:16
2.4–3.7
2.8–3.6
≤ 3.5%
Met (1 lot out)
Asp 329 deamidation
Peptide mapping
9:3
PTMs in adalimumab-aqvh similar or lower than adalimumab
Met
Asp 393 deamidation
N-term pyroGlu
Met 256 oxidation
Met 432 oxidation
Free SH
Ellman’s analysis
12:9
Free SH in adalimumab-aqvh similar or lower than adalimumab
Met
HCP, ppm
ELISA
18:17
HCP in adalimumab-aqvh lots similar or lower than adalimumab
Met
General properties
pI profile
icIEF
18:11
Adalimumab-aqvh main species pI similar to adalimumab
Met
Protein concentration, mg/mL
UV absorbance at 280 nm
36:16
46.9–51.9
46.0–49.6
45.3–50.3
Not met (2 lots out)e
SVPs, count/mLf (≥ 5 μm range)
MFI
23:16
3–816
93–1059
≤ 1837 particles/mL
Met
SVPs, count/mLf (≥ 10 μm range)
0–173
17–421
≤ 1053 particles/mL
Met
SVPs, count/mLf (≥ 25 μm range)
0–20
0–110
≤ 511 particles/mL
Met
Fab-associated biological activity
sTNF-α binding and neutralizationa
Apoptosis assay, % relative potency
26:16
92–106 (90% CI − 2.18 to 2.60)
94–106
EAC, ± 4.52
Met
sTNF-α binding by ELISA, % relative binding
17:16
87.8–109.8 (90% CI − 6.27 to 1.43)
80.6–107.5
EAC, ± 9.71
Met
sTNF-α Kd by SPR, Kd, pM
26:16
Comparable binding affinity (Kd) for adalimumab-aqvh and adalimumab
Met
mTNF-α binding
Cell-based mTNF-α binding by MSD, % relative binding
17:16
87.7–103.7
84.3–104.9
78.7–111.8
Met
Fab- and Fc-associated biological activity
ADCC activity
PBMC-mediated cell killing of mTNF-α-expressing cells, % relative activity
17:16
51.5–162.2
63.7–126.4
46.4–155
Met (1 lot out)
CDC activity
Complement-dependent cell killing of mTNF-α-expressing cells, % relative activity
17:16
86.3–114.1
96.9–114
88.7–119.3
Met (1 lot out)
Induction of regulatory macrophages
Inhibition of proliferation in Crohn’s MLR assay, % inhibition
8:9
Similar induction of regulatory macrophages for adalimumab-aqvh and adalimumab
aAcceptance criteria were dependent on the characteristic being analyzed. Where numerical acceptance criteria are specified, 90% of adalimumab-aqvh lots had to be within that quality range; for adalimumab-aqvh lots where N ≥ 10, acceptance criteria permitted one lot to lie outside the quality range. An exception to these criteria was for sTNF binding and neutralization, where criteria necessitated that the 90% CI for the difference in means fell within the equivalence acceptance criteria (EAC) based on the standard deviation of the results from the tested adalimumab lots
btGal: 7 of 17 lots out of range; however, the observed differences did not affect C1q, ADCC, or CDC activity
cSialic acid: 4 of 17 lots out of range
dnrCE-SDS: 9 of 16 lots out of range.
eProtein concentration: 2 of 16 lots out of range.
fNon-silicone droplet or air bubble, aspect ratio ≤ 0.85 for 5-, 10-, and 25-μm or more particles
gFcγRIIIa (158V) binding: 2 of 16 lots out of range; however, the observed binding difference did not affect similarity in FcγRIIIb or ADCC functional testing.
3.1 Primary Structure
Confirming primary structure is fundamental to the establishment of analytical similarity. The primary structures of representative adalimumab-aqvh and adalimumab lots were evaluated and confirmed using a reduced tryptic map with mass spectrometric analysis (Fig. 1a). The identity of each peptide was determined by mass spectrometry (MS). Corresponding peptides were found to have the same masses (within experimental error) in all adalimumab-aqvh and adalimumab lots tested. The sequence was confirmed with 100% coverage (Table 1). The primary structure was also confirmed by measurement of intact mass of representative adalimumab-aqvh and adalimumab lots (Table 1).
Fig. 1
The structural attributes of representative adalimumab-aqvh and adalimumab lots were compared using a tryptic map, b second-derivative FTIR spectra, c far UV CD, d near UV CD, and e differential scanning calorimetry. Cp heat capacity
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Correct disulfide structure is important for folding and functioning of a protein. The disulfide structures of adalimumab-aqvh and adalimumab lots were confirmed by non-reduced tryptic peptide maps with MS. The disulfide structure of adalimumab-aqvh matched the expected disulfide structure for immunoglobulin G (IgG) 1 and was the same as that of adalimumab (Table 1).
3.2 Higher Order Structure
To confirm similarity of the secondary structure, representative adalimumab-aqvh and adalimumab lots were analyzed by Fourier transform infrared (FTIR) spectroscopy. Second derivative spectra for adalimumab and adalimumab-aqvh showed a major amide I band at 1639 cm−1, indicating that β-sheet was the predominant secondary structure (Fig. 1b). Similar FTIR spectral features indicated that adalimumab and adalimumab-aqvh contained similar secondary structures. Circular dichroism (CD) was used to confirm similar secondary and tertiary structures between adalimumab-aqvh and adalimumab. Far ultraviolet (UV) CD spectra were visually similar for adalimumab and adalimumab-aqvh and were consistent with the expected predominantly β-sheet secondary structure (Fig. 1c). Near UV CD spectra were also highly similar, reflecting a consistently folded structure (Fig. 1d). Proteins typically exhibit intrinsic fluorescence arising from tryptophan residues and to a lesser extent tyrosine residues. The fluorescence spectrum reflects the tertiary structure of the protein, including the local environment of the aromatic amino acids and sometimes their configuration relative to one another. Fluorescence profiles and emission maxima were the same within experimental variability for adalimumab-aqvh and adalimumab (Table 1).
Differential scanning calorimetry (DSC) measures the heat capacity of a molecule as a function of temperature. The heat absorption profile reflects the energy required to disrupt intramolecular and intermolecular interactions and the temperature at which such structural unfolding takes place [thermal transition temperature (Tm)]. Therefore, DSC can be used to assess the similarity of higher-order structure. The adalimumab-aqvh and adalimumab samples showed three comparable thermal transitions typical of IgG: a poorly resolved transition at approximately 65 °C (Tm1, CH2 domain), followed by transitions at 72 °C (Tm2, Fab domain), and 82–83 °C (Tm3, CH3 domain) and highly similar DSC profiles (Fig. 1e).
3.3 Glycan Profile
Glycosylation plays an important role in the pharmacokinetics and effector functions of therapeutic monoclonal antibodies. Adalimumab has one N-linked glycosylation site on its Fc portion of the heavy chain (HC) for a total of two N-linked glycans per molecule. The N-glycan profiles of adalimumab-aqvh and adalimumab were visually similar, with some differences in the abundance of minor peaks (Fig. 2a). Glycans were also assessed quantitatively (Table 1). Levels of high-mannose glycans and total afucosylated glycans in adalimumab-aqvh were within the adalimumab average ± 3 σ for all but one lot (1 of 17), meeting the similarity criteria. The similarity criteria were not met for terminal galactose (tGal; seven of 17 lots) and sialic acid (4 of 17 lots) (Table 1).
Fig. 2
The purities of representative adalimumab-aqvh and adalimumab were compared using a labeled N-glycan profile, b size-exclusion UPLC, c nrCE–SDS, and d rCE–SDS. Fab fragment antigen binding, Fc fragment crystallizable, HL heavy–light, HHL heavy–heavy–light, HMWS high molecular weight species, LC, light chain, LMWS low molecular weight species, NGHC, non-glycosylated heavy chain
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Terminal galactosylation may affect C1q binding and CDC activity; however, the differences observed in the current study did not result in significant differences in CDC activity or C1q binding for these lots, and the similarity acceptance criteria were met for both functional attributes. High levels of sialylated Fc glycans have been associated with reduced ADCC activity [12]. However, the differences observed for the lots that did not meet the acceptance criterion were very small (< 0.2%) and did not affect ADCC because the ADCC activity of adalimumab-aqvh met similarity criteria. Therefore, adalimumab and adalimumab-aqvh were comparable for most of the glycan categories (high mannose, afucose, and total afucose), apart from tGal and sialic acid (Table 1). The observed differences in terminal Gal and sialic acid do not preclude a determination of similarity because these differences did not translate into functional differences when assessed by C1q, ADCC, and CDC activities.
3.4 Molar Mass and Self-Association
Size-exclusion chromatography with multi-angle light scattering (SEC–MALS), in combination with UV absorbance and refractive index detection, was used to characterize the size distributions of representative adalimumab-aqvh and adalimumab and to determine the molecular weight of the eluted peaks. The difference in the main peak molar masses from the theoretical mass of the monomer was < 2% for all lots tested. The main peak results for adalimumab-aqvh and adalimumab lots met the acceptance criteria and the high-molecular-weight species (HMWS) were similar (Table 1).
3.5 Purity
3.5.1 Size Variants
To determine any difference in size variants, the HMWS, monomer, and low-molecular-weight species (LMWS) of adalimumab-aqvh and adalimumab were separated and quantified by size exclusion–ultra-performance liquid chromatography (UPLC) (Fig. 2b). The amounts of HMWS and LMWS in adalimumab-aqvh lots were similar to or slightly lower than those in adalimumab samples, resulting in similar or slightly greater monomer relative areas in adalimumab-aqvh (Table 1).
Capillary electrophoresis in sodium dodecyl sulfate (CE–SDS) can also analyze size variants under denaturing conditions. Non-reduced CE–SDS (nrCE-SDS) measures covalently linked aggregates, partially reduced species, and fragments, in addition to the expected species with all disulfides formed (main peak). The nrCE–SDS profiles were qualitatively similar for adalimumab-aqvh and adalimumab (Fig. 2c). Although most lots met the acceptance criterion, some lots showed a slightly lower main peak relative area (Table 1). This small difference was primarily due to the presence of species resulting from incomplete disulfide bonds, such as light chain and heavy–heavy–light. These species are present only under denaturing conditions and did not result in a potency difference.
Reduced CE–SDS (rCE–SDS) detects non-glycosylated heavy chain and fragments, in addition to the expected light chain and heavy chain. rCE–SDS profiles were qualitatively similar between adalimumab-aqvh and adalimumab (Fig. 2d). Adalimumab-aqvh had a slightly lower percentage of nonglycosylated heavy chain than did adalimumab, resulting in a slightly higher percentage of light chain plus heavy chain for the adalimumab-aqvh samples. These differences represent <1% of molecules and are not expected to have any clinical impact.
Analytical ultra-centrifugation, which is orthogonal to SEC, assesses the size profile of a protein solution. Although it may be less sensitive for quantitation of minor species, it is free from the column interactions that can affect SEC measurements. The observed HMWS levels for adalimumab and for adalimumab-aqvh were all at or below the estimated method limit of quantification, indicating that the percentage of HMWS within the method capability of the samples was similar (Table 1).
3.5.2 Charge Variants
Cation-exchange chromatography (CEX) was used to evaluate the similarity of adalimumab-aqvh and adalimumab with respect to charge variants (Fig. 3a). The percentage of acidic peaks was similar between adalimumab-aqvh and adalimumab; just one adalimumab-aqvh lot was above the quality range for adalimumab (Table 1). The main peaks were higher and basic peaks were lower with adalimumab-aqvh than with adalimumab, primarily because of the presence of more lysine variants in adalimumab. Published literature [13] and additional internal studies with isolated fractions show no impact of C-terminal lysine variants on molecular function.
Fig. 3
The purity of representative adalimumab-aqvh and adalimumab were compared using a CEX and b icIEF. pI isoelectric point
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Imaged capillary isoelectric focusing was used to measure the isoelectric point (pI) of major and minor species. pI reflects the content of charged amino acids (and charged glycans, if present) and can be used to confirm molecular similarity. Charge variants monitored by imaged capillary isoelectric focusing (icIEF) are expected to include acidic peaks arising from deamidation and basic peaks arising from C-terminal lysine; other variants are possible. When comparing adalimumab-aqvh with adalimumab, the electropherography profiles were similar for the acidic species (Fig. 3b); however, the basic species were more abundant in adalimumab because of the higher number of C-terminal lysine variants (as shown in the CEX analyses).
3.5.3 Hydrophobic Variants
Reversed-phase chromatography separates product variants, including oxidized species, based on hydrophobicity. To improve resolution of minor species and facilitate comparison of adalimumab and adalimumab-aqvh, samples were treated with IgG-degrading enzyme of Streptococcus pyogenes (IdeS) protease and carboxypeptidase B (CPB), separating Fc and Fab domains and removing C-terminal lysine before chromatographic analysis. This method is used to quantify oxidized Fc variants because methionine oxidation in the Fc domain impacts neonatal Fc receptor (FcRn) binding and serum half-life [14]. Fc variants oxidized in adalimumab-aqvh were within the adalimumab average ± 3 σ for all but one lot (1 of 16), meeting the similarity criteria (Table 1).
3.5.4 Post-Translational Modifications
Reduced tryptic peptide mapping with liquid chromatography–mass spectrometry (LC–MS) was used to assess specific stability-indicating post-translational modifications. Using selected ion monitoring, peak areas of modified peptides were compared with the total area for that peptide. Levels of all five monitored modifications were of similar magnitude in adalimumab-aqvh and adalimumab (Table 1).
3.5.5 Free Thiol
Levels of free thiol in the samples were determined using the Ellman reagent. It was previously confirmed that adalimumab-aqvh and adalimumab contain all expected intra-chain and inter-chain disulfide bonds. The free thiol content in adalimumab-aqvh was similar to but slightly lower than that in adalimumab, which met the similarity acceptance criterion (Table 1).
3.5.6 Host-Cell Proteins
An enzyme-linked immunosorbent assay (ELISA) was used to analyze the host-cell protein (HCP) content of adalimumab-aqvh and adalimumab. HCP levels in adalimumab-aqvh were less than the limit of quantification (≤ 0.2 ppm). HCP levels in adalimumab were still low, at approximately 1–6 ppm, but consistently quantifiable and at least a few fold higher than those in adalimumab-aqvh. These data indicated that adalimumab-aqvh contained lower HCP levels than adalimumab (Table 1).
3.6 General Properties
Protein concentrations of adalimumab-aqvh and adalimumab were compared using absorbance at 280 nm and an experimentally determined extinction coefficient of 1.45 mg/mL·cm. The acceptance criterion was not met with two adalimumab-aqvh lots outside of the quality range based on mean ± 3 σ of adalimumab lots. However, all 14 of the adalimumab-aqvh lots manufactured after a process improvement were within the quality range (Table 1).
Subvisible particles, which can include potentially immunogenic protein particles, were evaluated using microflow imaging (MFI). To minimize the contribution of spherical silicone oil droplets, which typically have an aspect ratio ≥ 0.85, a morphologic filter was applied to report non-spherical particles with aspect ratio < 0.85. The results indicated that adalimumab-aqvh contained a similar number of or fewer non-spherical particles than adalimumab at size ranges of ≥ 5 μm (Table 1).
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3.7 Fab-Associated Biological Activity
Binding of soluble TNF-α (sTNF-α) to its receptor TNFR1 on immune cells results in pro-inflammatory activities. Adalimumab inhibits sTNF-α–mediated activities via its primary mechanism of action which is through binding to, and neutralizing sTNF-α. An sTNF-α-indirect ELISA assay was used to compare binding of adalimumab and adalimumab-aqvh. The results were presented as relative binding activity against a primary reference standard of adalimumab-aqvh. The 90% CI for the difference in means fell within the equivalence acceptance criteria derived from adalimumab results for sTNF-α binding activity (Table 1), demonstrating that binding to sTNF-α was similar for adalimumab and adalimumab-aqvh (Fig. 4a). A surface plasmon resonance (SPR)-based orthogonal method was also used to characterize the sTNF-α binding affinity (KD) of adalimumab and adalimumab-aqvh. The association constant (ka) and the dissociation rate constant (kd) were comparable between adalimumab-aqvh and adalimumab lots (Fig. 4b).
Fig. 4
Fab (antigen-binding)-associated biological activity of representative adalimumab-aqvh and adalimumab were compared by assessing a sTNF-α binding activity by indirect ELISA, b isoaffinity plot showing the association rate constant and the dissociation rate constant by SPR, c sTNF-α neutralization potency, and d transmembrane-bound TNF-α binding by MSD assay. *Indicates average value from two accepted runs. ka association rate constant, kd dissociation rate constant, TNF-α tumor necrosis factor alpha
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sTNF-α neutralization potency was determined by a cell-based assay that measures the ability of adalimumab to bind to TNF-α and inhibit sTNF-α–induced apoptosis. The results are presented as mean potency relative to a primary reference standard. The 90% CI for the difference in means fell within the equivalence acceptance criteria of ± 1.5 σ based on the standard deviations calculated from the adalimumab lots (Table 1), indicating that the ability of adalimumab-aqvh and adalimumab to neutralize sTNF-α activity was similar (Fig. 4c).
Binding to membrane-associated TNF-α (mTNF-α) was also measured because adalimumab mediates Fab- and Fc-dependent activities by binding to mTNF-α. Using a meso-scale discovery (MSD) electrochemiluminescence assay, binding of adalimumab-aqvh and adalimumab to mTNF-α (Fig. 4d) was similar, and overall similarity was met between adalimumab and adalimumab-aqvh lots (Table 1).
3.8 Fab- and Fc-Associated Biological Activity
ADCC by adalimumab is mediated by cross-linking of Fc gamma receptors, such as FcγRIIIa, on the surface of immune effector cells and binding to transmembrane TNF-α. A physiologically relevant assay was established to measure human donor derived peripheral blood mononuclear cell (PBMC)-mediated cell killing of adalimumab- and adalimumab-aqvh-bound mTNF-α cells. Based on the ADCC activity results (Fig. 5a), adalimumab-aqvh met the criteria of similarity to adalimumab (Table 1). Although one adalimumab-aqvh lot was slightly above the similarity range of ADCC (relative ADCC activity: ~ 162%; acceptance criteria: 46–155%), these results were within the accepted assay variability.
Fig. 5
Fab- and Fc-associated biological activity of representative adalimumab-aqvh and adalimumab lots were compared using a relative ADCC activity by PBMC-mediated cell killing of mTNF-α-expressing cells, b relative CDC activity using mTNF-α-expressing cells, and c the induction of regulatory macrophage activity in MLR assays. ADCC antibody dependent cell cytotoxicity, CDC complement-dependent cytotoxicity
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Adalimumab also depletes cells that express mTNF-α by complement fixation on the antibody- coated target cells. CDC can be triggered by classic and alternate complement pathways through complement proteins such as C1q and C3a. CDC is a physiologically relevant assay to interrogate this mechanism of action and to assess the impact of any structural attributes that may influence binding to mTNF-α or complement proteins. The assay results showed similarity of CDC activity between adalimumab-aqvh and adalimumab (Fig. 5b, Table 1). One lot was slightly below the similarity range of CDC activity, but there was no effect on binding to complement protein C1q (which is the first step in the activation of the classic CDC pathway) for this lot (Fig. 6a). The results for CDC activity further showed that slight variations in tGal levels in adalimumab-aqvh did not affect CDC activity, compared with the adalimumab lots. These observations are in line with published reports [15], indicating that incremental changes in tGal levels in antibodies show a subtle effect in CDC activity.
Fig. 6
Fc-associated biological activity of representative adalimumab-aqvh and adalimumab lots were compared using a complement component 1q by ELISA, b FcγRIIIa 158V relative binding affinity by SPR, c FcγRIIIb relative binding affinity by SPR, and d FcRn relative binding affinity by SPR. C1q complement component 1q, FcγRIIIa Fc gamma receptor IIIa 158V, FcγRIIIb Fc gamma receptor IIIb, FcRn neonatal Fc receptor
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Anti-TNF antibodies have been shown to inhibit T-cell proliferation by inducing regulatory macrophages in both a Fab- and Fc-dependent manner. The mixed lymphocyte reaction (MLR) assay is used to evaluate T-cell proliferation in response to activation by allogeneic stimulator cells. With the MLR assay, adalimumab and adalimumab-aqvh showed similar anti-proliferative activity against activated lymphocytes (Fig. 5c).
3.9 Fc-Associated Biological Activity
A C1q ELISA was used to test C1q binding, representing the first step in initiation of the classic CDC pathway for target-bound adalimumab. As mentioned previously herein, the C1q binding results (Fig. 6a) showed similar binding between adalimumab-aqvh and adalimumab (Table 1). Relative binding affinity to FcγRIIIa (158V) and FcγRIIIb was comparable for adalimumab and adalimumab-aqvh lots (Fig. 6b, c). Similarity criteria were met for FcγRIIIb, whereas 2 of 16 adalimumab-aqvh lots that were tested showed slightly lower relative binding affinity outside the three standard deviation range of adalimumab for FcγRIIIa (158V) (Table 1). These slight differences in relative binding affinity did not result in any impact on similarity in ADCC activity.
Binding, followed by recycling, via FcRn is a known mechanism in maintaining the homeostasis of IgG molecules. An SPR-based FcRn binding assay was used to compare FcRn binding responses of adalimumab-aqvh and adalimumab. The results showed similar binding affinity between adalimumab-aqvh and adalimumab (Fig. 6d).
3.10 Forced Degradation Under Heat Stress
With heat exposure at 40 °C, similar degradation profiles for adalimumab-aqvh and adalimumab were observed, with small increases in HMWS and in LMWS detected by SE–UPLC (Fig. 7a). Similar degradation profiles were also observed by CEX after CPB treatment. The major degradation pathway observed for both was attributed to deamidation as indicated by an increase in the number of acidic peaks. Smaller increases in CEX basic peaks were observed (Fig. 7b).
Fig. 7
The degradation profiles under heat exposure at 40 °C of representative adalimumab-aqvh and adalimumab lots were compared using a size-exclusion UPLC and b CEX with carboxypeptidase B treatment. HMWS high-molecular-weight species, LMWS low-molecular-weight species, M months, T time
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4 Discussion
The totality of structural, functional, and stability data in this comparative analytical assessment supports a conclusion of biosimilarity between adalimumab-aqvh and the reference product, adalimumab. The structural data showed that adalimumab-aqvh and adalimumab have identical amino acid sequences and disulfide structure. Adalimumab-aqvh met the similarity criteria for most of the glycan categories (high mannose, afucose, and total afucose), with the exception of tGal and sialic acid. The observed differences in tGal and sialic acid do not preclude a determination of similarity because these differences did not translate into functional differences as assessed by C1q, ADCC, and CDC activities. Adalimumab-aqvh met similarity criteria for pI, and molecular weight (by SEC–MALS). Adalimumab-aqvh did not meet similarity criteria to adalimumab for protein concentration because two lots had slightly higher values. However, after a process improvement, all subsequent lots were within the similarity range for protein concentration.
Adalimumab-aqvh met quantitative similarity criteria for size variants by size-exclusion UPLC and rCE–SDS, charge variants by CEX, Fc oxidation by reversed-phase (RP) high performance liquid chromatography (HPLC), and subvisible particles by MFI. Adalimumab-aqvh did not meet the criteria for percentage main peak by nrCE–SDS, however the differences observed had no impact on potency or stability. Adalimumab-aqvh met all similarity criteria for all primary (known) mechanism of action assays and for all likely and plausible mechanism of action assays, with the exception of FcγRIIIa binding, for which two lots were slightly outside of similarity criteria. These differences in FcγRIIIa binding did not affect similarity in ADCC activity, a plausible mechanism of action mediated by activated FcγRIIIa (CD16a). The differences in FcγRIIIa binding also did not affect the pharmacokinetics or safety of adalimumab-aqvh when evaluated in the clinical trial in which biosimilarity of adalimumab-aqvh to adalimumab was assessed [9].
Additionally, functional characterization using orthogonal assays for known, likely, and plausible mechanisms of action further supported similarity between adalimumab-aqvh and adalimumab. Adalimumab-aqvh and adalimumab exhibited comparable degradation profiles under stressed storage conditions.
5 Conclusions
Overall, the results of this comparative analytical assessment showed that adalimumab-aqvh is highly similar to adalimumab. Similarity was shown in the biological activities related to the known, likely, and plausible mechanism of action of adalimumab. Minor differences in physicochemical attributes did not affect functional activity, and, relatedly, should not affect the clinical efficacy or safety of adalimumab-aqvh. The similarities between adalimumab-aqvh and adalimumab predict similar efficacy and safety outcomes in clinical trials, which was confirmed by the pharmacokinetic, safety, immunogenicity, and efficacy clinical trials that have been conducted [9].
Acknowledgements
The authors acknowledge the technical contributions of Andy Thuy, Zahidul Islam, Shruti Sahay, Noah Luther, Michelle Stanfill, Justin Brown, Matt McQueen, Dimitri Diaz, Ben Andrews, Greg East, Stephanie Desanti, Claire Willey, Shannon Williams, Aditya Gandhi, Anton Karnoup, and Eun Ji Joo. Medical writing and editorial assistance was provided by Chantel Kowalchuk, PhD, and Kim Enfield, PhD, (ApotheCom, San Francisco, CA). The funding for this study and medical writing support was provided by Coherus BioSciences. (Redwood City, CA).
Declarations
Funding
This study was funded by Coherus BioSciences, Inc., Redwood City, CA.
Conflict of interest
All authors, except TA, are employees and are stockholders of Coherus BioSciences. At the time of the study, all authors were employees of Coherus Biociences, Inc.
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Data availability statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability
Not applicable.
Author contributions
The main text of the paper was written by Yijia Jiang, Taruna Arora, Scott Klakamp, Janice Davis, Yasmin A. Chandrasekher, and Karen Miller. All authors contributed to the collection and analysis of data. All authors reviewed and approved the final manuscript. Medical writing and editorial assistance was provided by Chantel Kowalchuk, PhD, and Kim Enfield, PhD, (ApotheCom, San Francisco, CA).
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