The disfunction or deficiency of the C1 esterase inhibitor (C1INH) is associated with hereditary or acquired angioedema (HAE/AAE), a rare life-threatening condition characterized by swelling in the skin, respiratory and gastrointestinal tracts. The current treatment options may carry the risks of either viral infection (plasma-derived Berinert^®) or immune reaction (human recombinant C1INH from rabbit milk, Ruconest^®). This study describes the physicochemical and biological characterization of a novel recombinant human C1 esterase inhibitor (rhC1INH) from Chinese hamster ovary (CHO) cells for the treatment of hereditary angioedema compared to the marketed products Berinert^® and Ruconest^®. The mass spectrometry results of total deglycosylated rhC1INH revealed a protein with a molecular mass of 52,846 Da. Almost full sequence coverage (98.6%) by nanoLC-MS/MS peptide mapping was achieved. The purity and C1s inhibitory activity of rhC1INH from CHO cells are comparable with Ruconest^®, although we found differences in charge isoforms distribution, intact mass values, and N-glycans profile. Comparison of the specific activity (IC₅₀ value) of the rhC1INH with human C1 esterase inhibitor from blood serum showed similar inhibitory properties. These data allow us to conclude that the novel rhC1INH molecule could become a potential therapeutic option for patients with HAE/AAE.

INTRODUCTION. Bioanalytical techniques are characterised by greater variability and lower stability than physicochemical methods because live test systems are inherently labile. Since regulatory standards do not establish a unified approach, the selection of system suitability criteria and acceptance criteria for test results is based on validation studies.AIM. This study aimed to validate an analytical procedure for evaluating the biological activity of a medicinal product based on the investigational tocilizumab biosimilar GNR-087 and determine the quantitative limits for system suitability criteria and acceptance criteria for test results.MATERIALS AND METHODS. The biological activity of the investigational tocilizumab biosimilar was assessed by the inhibition of IL-6-induced secreted embryonic alkaline phosphatase (SEAP) expression by HEK-Blue™ IL-6 cells. Statistical processing of the obtained results was performed using Prism 6.0 software.RESULTS. The specificity of the analytical procedure was confirmed by the dose-dependent inhibition of IL-6-induced SEAP expression by cells observed with tocilizumab. The analytical procedure was linear, with a coefficient of determination R²≥ 0.99. The precision of the analytical procedure was satisfactory; its repeat ability varied from 2 to 9%, and its intermediate precision was 14%. The recovery coefficients (Rc) for spiked samples simulating activity levels of 60–140%, including blinded samples, ranged from 80 to 120%. The theoretical values of relative potency (RP) were within the confidence intervals of the mean relative potency estimates, which confirmed the accuracy of the analytical procedure. The validation confirmed the robustness of the analytical procedure to controlled variations, including the use of reporter cells at different passages (with a coefficient of variation for relative potency estimates (CV_RP) of 10%), different IL-6 lots (with a CV_RP of 1%), and different SEAP detection reagent lots (with a CV_RP of 3%); the Rc remained in the range of 80–120% of the nominal RP value.CONCLUSIONS. The analytical procedure for evaluating the biological activity of the investigational tocilizumab biosimilar meets the validation criteria, including accuracy, linearity, precision, specificity, and robustness. The study established system suitability criteria and acceptable limits for biological assay results. This analytical procedure can be used both for routine biological activity control and for demonstrating the biosimilarity of new medicinal products to the original (reference) tocilizumab-based medicinal product.

Background/Objectives: Nusinersen is a synthetic antisense RNA oligonucleotide employed in the management of spinal muscular atrophy, a rare neuromuscular disorder, by modulating the alternative splicing of the survival motor neuron 2 (SMN2) gene. GNR-100 represents the first generic version of the reference listed drug (RLD), containing nusinersen sodium as the active pharmaceutical ingredient. We performed comprehensive evaluations in accordance with FDA guidelines, including side-by-side comparative analyses of critical quality attributes, to thoroughly characterize the structural and functional properties of both nusinersen products. Results/Methods: GNR-100 was comprehensively demonstrated to be highly similar to RLD in terms of oligonucleotide structure, physicochemical properties, impurity profile, and in vitro cell-based assays for SMN-gene splice-switching and SMN-protein activity. Structural analyses confirmed that the oligonucleotide primary sequences and chemical structures were identical. The diastereomeric composition and higher-order structures were also similar between the proposed generic and the reference product. Comparable resistance to phosphodiesterase degradation and nearly identical melting temperatures of the oligonucleotide duplexes with their complementary strand further substantiated the structural sameness of the nusinersen products. The impurity profile of the proposed therapeutic oligonucleotide was consistent with that of RLD, and the collectively reduced levels of impurities, as assessed by orthogonal analytical methods, indicated no meaningful impact on the safety profile. Moreover, both products exhibited comparable biological activity in enhancing the production of full-length SMN2 mRNA transcripts and functional SMN protein in fibroblasts derived from SMA patients. Conclusions: These quality studies demonstrate that GNR-100 exhibits no significant differences from the licensed drug across structural, physicochemical, biophysical, and biological attributes, establishing its potential as a cost-effective therapeutic alternative for patients with spinal muscular atrophy.

Idursulfase and idursulfase beta currently used for enzyme replacement therapy of mucopolysaccharidosis type II (MPS II) do not cross the blood-brain barrier (BBB), which significantly limits the treatment of neurocognitive impairment in patients. Verenafusp alfa (Clotilia®, GENERIUM JSC) contains a recombinant modified iduronate-2-sulfatase enzyme covalently linked to the Fab fragment of a monoclonal antibody against the human insulin receptor, which crosses the BBB. Objective: The aim of this multicenter, open-label, multicohort phase II–III study was to evaluate the safety, pharmacokinetics, pharmacodynamics (PD), efficacy, and optimal dose selection of verenafusp alfa in different age groups of patients. Materials and methods: The interim analysis included data from 18 patients under 18 years of age in 6 age cohorts. Cohort enrollment was performed sequentially with decreasing patient age, first for the 2 mg/kg dose (Group 1) in cohorts 2, 3, 5, and then for the 3 mg/kg dose (Group 2) in cohorts 4, 6, 7. The study included 52 weekly infusions of verenafusp alfa. The PD criterion was the level of glycosaminoglycans (GAGs) in urine, blood, and cerebrospinal fluid (CSF). Efficacy parameters included assessment of somatic manifestations and neurocognitive functions; safety parameters included assessment of adverse events (AEs) and the dynamics of anti-drug antibody (ADA) formation. Results: The increase in verenafusp alfa concentration in CSF was more pronounced in Group 2 (3 mg/kg) – 252.5 [221.4; 351.54] pg/mL, as well as in the group of children under 6 years of age – 302.02 [244.72; 388.83] pg/mL. The decrease in heparan sulfate (HS) levels in CSF at week 52 was more pronounced in Group 2 (3 mg/kg) – 49% (p=0.084), and also in the group of patients under 6 years of age – 53% (p=0.027). Urinary and blood GAG levels remained stable, taking into account prior idursulfase therapy in 94% of patients. An 18% increase in the 6-minute walk test distance was observed in the overall group of patients under 18 years of age (p=0.020), accompanied by a statistically significant increase in range of motion in the shoulder, knee, and left hip joints. Liver and spleen volumes remained stable. Stabilization and/or improvement of neurocognitive function parameters were observed in patients with MPS II. The AEs observed in 50% of patients were non-serious, classified as infusion reactions of mild or moderate severity, with a predictable and manageable spectrum and frequency. ADA formation against the drug was observed in 40% of patients. Conclusion: Weekly intravenous administration of verenafusp alfa for one year achieved control of urinary GAG levels, stabilization and/or improvement of neurocognitive functions, and reduction of somatic manifestations of MPS II in previously treated patients with idursulfase drugs aged 2 to 16 years. After one year of therapy, an increase in verenafusp alfa concentration and a decrease in HS levels in CSF were observed, confirming its ability to cross the BBB and prevent neurodegenerative changes in the CNS.

INTRODUCTION. Romiplostim treatment of patients with idiopathic thrombocytopenic purpura is associated with formation of anti-drug antibodies (ADA), which often leads to the serious adverse events. A method based on biolayer interferometry is proposed for determination of binding (total) ADA (bADA) to romiplostim in human serum. From a technological point of view, instruments using this principle have some advantages (higher throughput, prolonged service intervals, low level of equipment contamination during analysis of biosamples) over those based on surface plasmon resonance, which are used to monitor bADA levels during clinical trials of the original drug. The method should be validated to ensure reproducibility of measurements and standardise clinical laboratory trials of the immunogenicity of a biosimilar drug.AIM. To establish the key validation characteristics of a biolayer interferometry-based method for the determination of total anti-drug antibodies to romiplostim in human serum.MATERIALS AND METHODS. The method is used to detect the specific interaction of binding antibodies with biotinylated romiplostim immobilised on the streptavidin-coated biosensors. The method includes a screening test to determine bADA presence or absence in the samples; a confirmatory test to check the specificity of bADA binding to romiplostim; determination of antibody titer and maximal dilution, that allows to detect bADA in the samples. Positive control samples containing different concentrations of polyclonal rabbit antibodies to romiplostim were used in the work. Detection of protein complex formation was performed in real time mode using an Octet® QKe interferometer.RESULTS.  The biological variability factor and the limit of not significant inhibition were 1.481 and 34.2%, respectively. The precision and specificity of the method were confirmed. The lower limit of bADA detection in the absence of romiplostim was 622 ng/mL. The analytical signal of 8 out of 10 blood serum samples after the addition of bADA increased by more than 1.481 times when assessing the matrix effect in the screening test; the percentage inhibition value exceeded 34.2% when analysing 8 out of 10 samples with the addition of bADA in the confirmatory test. There was no high-dose effect at bADA concentrations from 0.8 to 50 μg/mL. The results of bADA determination in the screening test were robust to the presence of 1 ng/mL romiplostim in the samples. Streptavidin-coated biosensors with immobilised romiplostim maintained stability after 14 days of storage at a temperature of (5±3) °С and at least 20 regeneration cycles.CONCLUSIONS. Analysis of the validation results obtained confirms the suitability of the method for reliable and reproducible determination of binding (total) antibodies to romiplostim in human serum.