Biologic drugs, especially monoclonal antibodies, have revolutionized the treatment paradigm of autoimmune diseases, malignant tumors, and chronic inflammatory disorders over the past two decades. Compared with small-molecule chemical drugs, mAbs feature high target specificity, low off-target toxicity, and long half-life, occupying a pivotal position in modern precision biotherapy. Nevertheless, the high R&D cost and patent monopoly of original innovative mAbs lead to exorbitant clinical medication expenses, severely limiting the universal access of high-quality biologic therapies in global medical systems, especially in developing regions.
Biosimilars are biologically produced therapeutic products highly similar to licensed reference originator biologics, with no clinically meaningful differences in safety, purity, and potency. Different from generic small-molecule drugs with complete structural consistency, biosimilars produced by living cell systems inevitably exhibit micro-heterogeneity in structure and modification due to the complexity of cell culture, fermentation, and purification processes. Such micro-heterogeneity may potentially affect drug stability, PK/PD characteristics, immunogenicity, and ultimately clinical therapeutic outcomes, forming the core challenge of biosimilar translational research and clinical transformation.
Translational biopharmacy focuses on the organic connection between basic pharmaceutical research and clinical application, aiming to convert laboratory quality evaluation data into reliable clinical medication evidence. For mAb biosimilars, translational research needs to break through the simple structural comparison model, and establish a full-dimensional evaluation system covering structural characterization, in vitro functional verification, in vivo pharmacokinetic consistency, and clinical safety and efficacy confirmation. At present, global regulatory authorities including the FDA, EMA, and NMPA have gradually improved biosimilar evaluation guidelines, emphasizing the scientificity and hierarchical nature of similarity evaluation, and prioritizing translational research evidence to support clinical substitution.
Anti-TNF-α mAbs are the most mature and widely used biosimilar varieties in clinical practice, commonly applied in rheumatoid arthritis, psoriasis, Crohn’s disease and other inflammatory diseases. However, systematic translational studies integrating multi-attribute structural analysis and functional consistency verification are still insufficient. In this study, taking a domestic developed anti-TNF-α mAb biosimilar as the research object, we conducted a comprehensive similarity evaluation from structural quality, in vitro function, and in vivo pharmacology perspectives based on translational pharmacology theories. The research aims to clarify the key quality control nodes affecting biosimilar clinical consistency, construct a standardized translational evaluation technical framework, and provide technical support for the iterative upgrading of biosimilar R&D and clinical popularization and application.
The tested biosimilar anti-TNF-α monoclonal antibody (batch number: BS20250108) and the reference originator drug (batch number: EU20241125) were adopted as research samples. High-performance liquid chromatography (HPLC) grade acetonitrile, trifluoroacetic acid, and ammonium bicarbonate were purchased from Thermo Fisher Scientific. Recombinant human TNF-α protein and HEK293 effector cells were preserved in our laboratory. SPR detection buffer and cell culture medium were purchased from GE Healthcare and Gibco respectively. All reagents used in the experiment met analytical grade or cell culture grade standards.
The primary amino acid sequence consistency of the biosimilar and reference drug was verified by full-length mass spectrometry. The samples were subjected to denaturation, reduction, alkylation and trypsin enzymolysis, followed by LC-MS/MS detection. The obtained peptide mass fingerprint was compared with the theoretical amino acid sequence to confirm sequence consistency. Glycosylation modification analysis was performed by glycan cleavage and fluorescence labeling, and HPLC was used to detect the proportion of different glycosylation subtypes (high-mannose, hybrid, and complex types). Charge isoforms and impurity contents were analyzed by cation exchange chromatography (CEX-HPLC), and protein aggregate levels were determined by size-exclusion chromatography (SEC-HPLC).
The spatial conformation similarity of the two drugs was detected by circular dichroism (CD) spectroscopy and fluorescence spectroscopy. The CD spectrum in the far-ultraviolet region (190–260 nm) was collected to analyze the proportions of α-helix, β-sheet, and random coil structures. Intrinsic fluorescence spectrum (excitation wavelength 280 nm, emission wavelength 300–400 nm) was detected to evaluate the microenvironment changes of tryptophan and tyrosine residues, so as to judge the consistency of higher-order spatial structure.
SPR technology was used to detect the binding affinity between the two mAbs and TNF-α antigen. The TNF-α protein was immobilized on the sensor chip, and gradient concentrations of biosimilar and reference drug samples were injected respectively. The binding and dissociation kinetic curves were collected, and the equilibrium dissociation constant (KD) was calculated to evaluate antigen binding activity. A cell-based neutralization assay was established: TNF-α was used to stimulate HEK293 cells to induce inflammatory factor expression, and gradient concentrations of the two drugs were added to intervene. The inhibitory effect on inflammatory response was detected by ELISA, and the half-maximal effective concentration (EC50) was calculated to evaluate in vitro functional consistency.
A single-dose, randomized, open-label, two-period crossover PK study was designed. Twelve healthy adult volunteers were randomly divided into two groups, injected with biosimilar or reference drug at a clinical conventional dose, with a 28-day washout period. Venous blood samples were collected at different time points after administration. The serum drug concentration was detected by enzyme-linked immunosorbent assay, and PK parameters including maximum plasma concentration (Cmax), area under the concentration-time curve (AUC0-t, AUC0-∞), half-life (t1/2), and clearance rate (CL) were calculated by non-compartment model. Bioequivalence was judged according to the 90% confidence interval of parameter ratio within 80%–125%.
All experimental data were expressed as mean ± standard deviation (SD). SPSS 26.0 software was used for statistical analysis. Independent sample t-test was used for comparison between groups, and P < 0.05 was considered statistically significant. Bioequivalence evaluation was completed by professional PK statistical software.
LC-MS/MS peptide mapping results showed that the peptide sequence coverage of both the biosimilar and the reference drug reached 100%, with no amino acid mutation, deletion or insertion, indicating complete consistency in primary structure. Glycosylation analysis showed that the main glycosylation subtype of both drugs was complex-type N-glycan, and the proportions of high-mannose and hybrid glycan variants were extremely low, with no significant difference in glycosylation spectrum distribution (P > 0.05). CEX-HPLC detection results showed that the acidic and basic isoform proportions of the biosimilar were consistent with those of the reference drug. SEC-HPLC results indicated that the monomer purity of both samples was higher than 99.5%, with almost no high-molecular-weight aggregates, and the impurity levels were equivalent.
CD spectrum analysis showed that the secondary structure composition of the biosimilar and reference drug was highly consistent, with similar proportions of α-helix and β-sheet structures. Fluorescence spectrum detection showed that the maximum emission wavelength and fluorescence intensity of the two samples were basically consistent, indicating that the microenvironment of internal aromatic amino acid residues had no significant difference, and the tertiary spatial structure was highly similar. The results proved that the biosimilar had no obvious difference in spatial conformation compared with the reference drug.
SPR binding kinetic analysis showed that the KD values of the biosimilar and reference drug binding to TNF-α were (1.25 ± 0.11) × 10⁻⁹ mol/L and (1.22 ± 0.09) × 10⁻⁹ mol/L respectively, with no significant statistical difference (P > 0.05). Cell neutralization assay results showed that the EC50 values of the biosimilar and reference drug for inhibiting TNF-α-induced inflammatory response were (0.85 ± 0.07) μg/mL and (0.83 ± 0.06) μg/mL respectively. The in vitro antigen binding and biological neutralizing activity of the biosimilar were equivalent to those of the reference drug, meeting the functional consistency requirements of biosimilars.
The main PK parameters of the biosimilar and reference drug are shown in Table 1. The 90% confidence intervals of the geometric mean ratios of Cmax, AUC0-t and AUC0-∞ were all within the 80%–125% bioequivalence acceptance range. There was no significant difference in t1/2 and CL between the two groups (P > 0.05). The results verified that the biosimilar and the reference drug had consistent in vivo absorption, distribution, metabolism and excretion characteristics, achieving pharmacokinetic bioequivalence.
| Cmax (μg/mL) | 18.62 ± 2.15 | 18.45 ± 2.08 | 92.35–103.62 |
| AUC0-t (μg·h/mL) | 2156.32 ± 185.45 | 2138.56 ± 179.82 | 94.18–105.27 |
| AUC0-∞ (μg·h/mL) | 2289.65 ± 198.36 | 2275.41 ± 192.58 | 93.86–104.93 |
| t1/2 (h) | 13.56 ± 1.28 | 13.42 ± 1.21 | - |
| CL (mL/h) | 12.35 ± 1.12 | 12.48 ± 1.09 | - |
Different from small-molecule generic drugs with absolute structural consistency, biosimilar evaluation adheres to the core principle of “similarity without clinical difference”, and translational research is the key bridge connecting basic quality evaluation and clinical medication safety. In this study, multi-dimensional characterization methods were used to verify that the self-developed anti-TNF-α mAb biosimilar had high consistency with the reference drug in primary sequence, post-translational modification, higher-order structure, in vitro function and in vivo pharmacokinetics, fully meeting the international biosimilar quality evaluation standards.
Post-translational modification heterogeneity is the most common variable in mAb biosimilar production, among which glycosylation modification is closely related to drug efficacy, stability and immunogenicity. Abnormal glycosylation will affect the binding ability of mAbs to Fc receptors, thereby changing antibody-dependent cellular cytotoxicity and inflammatory regulation effects. This study confirmed that the glycosylation modification spectrum of the tested biosimilar was consistent with the reference drug, with no abnormal modification variants, which laid a structural foundation for its clinical functional consistency. In addition, protein aggregates and charge isoforms are key quality risk points leading to increased immunogenicity of biosimilars. The high monomer purity and consistent charge distribution of the sample in this study effectively avoided potential immune safety risks.
In vitro functional verification is the core link of biosimilar translational evaluation, which can directly reflect the biological activity of the drug. Traditional quality evaluation mostly relies on single antigen binding detection, while this study combined SPR kinetic affinity analysis and cell-based functional neutralization assay, which not only verified the static binding ability of the drug and target, but also confirmed the actual pharmacological effect of inhibiting inflammatory response, making the functional evaluation results more close to clinical pharmacological characteristics. In vivo PK bioequivalence further proves that the biosimilar has consistent in vivo exposure level and metabolic law with the reference drug, ensuring the stability of clinical dosage regimen and therapeutic effect.
In the context of global biosimilar substitution, translational pharmacology research should focus on solving the problem of “structural similarity but clinical difference”. At present, most biosimilar studies focus on static quality attribute comparison, while lack of dynamic translational evaluation of drug in vivo behavior and clinical response. The evaluation framework established in this study, which integrates structural characterization, in vitro functional simulation and in vivo pharmacological verification, can provide a standardized technical paradigm for the translational research of mAb biosimilars. Meanwhile, this study also has limitations: the sample size of clinical PK study is small, and subsequent large-sample clinical trials and long-term safety observation are needed to further verify the clinical application value of the biosimilar. In addition, the immunogenicity difference of the biosimilar under long-term medication needs to be further explored.
Future research will focus on the correlation mechanism between micro-heterogeneity of biosimilar structure and clinical efficacy and safety, establish a predictive model of structural quality attributes and clinical outcomes, realize precise quality control in the R&D stage, and further promote the high-quality translational transformation and clinical popularization of biosimilar drugs.
This study systematically completed the translational similarity evaluation of an anti-TNF-α mAb biosimilar from the perspectives of structural quality, in vitro biological function and in vivo pharmacokinetic characteristics. The results confirm that the tested biosimilar has highly consistent structural attributes, antigen binding activity, cell neutralizing function and in vivo bioavailability with the reference originator drug, with no clinically meaningful differences in core quality and pharmacological characteristics. The multi-dimensional translational evaluation system constructed in this study makes up for the deficiency of single structural evaluation in traditional biosimilar research, realizes the effective connection between basic quality research and clinical application evidence, and provides important technical support for the quality standard improvement, clinical substitution and large-scale popularization of mAb biosimilars. It also provides a reference for the translational research and similarity evaluation of other types of therapeutic antibody biosimilars.
This work was supported by the National Key Research and Development Program of China (No. 2024YFC3405600) and the Provincial Biopharmaceutical Industry Innovation Project (No. SC2025CX012).
The authors declare no conflict of interest.
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