Background. Currently, the main treatment for Gaucher disease is enzyme replacement therapy. Recombinant glucocerebrosidase (imiglu-cerase) is the first biotechnological drug for enzyme replacement therapy with proven clinical efficacy and safety for the treatment of patients of different ages with Gaucher disease type 1 and type 3, used in clinical practice since 1994. In Russia, within the framework of the “Pharma 2020” pharmaceutical industry development strategy, the first biosimilar of imiglucerase, the drug Glurazyme®, was developed. The obtained results of preclinical studies became the basis for a phase I randomized comparative crossover clinical trial.The objective of the study was to assess the short-term safety and pharmacokinetic parameters of Glurazyme® in comparison with the Cerezyme® after a single intravenous administration to healthy volunteers.Materials and methods. 23 healthy volunteers aged 18—45 years were included in a 3-stage clinical trial. The study during the 1st and 2nd stages was open, randomized, comparative, crossover. At the 1st stage, volunteers from the 1st group received the Glurazyme®, from the 2nd group — the Cerezyme® once in doses of 30 U/kg. At the 2nd stage, Cerezyme® was administered to the 1st group, Glurazyme® - to the 2nd group once at doses of 30 U/kg. After the end of the 1st and 2nd stages, the 3rd stage was carried out for the 3rd group (n = 5) with the administration of the test drug once at a dose of 60 U/kg.Results. For all studied pharmacokinetic parameters, after administration of the test and reference drugs in doses of 30 U/kg, 90 % confidence interval was in the range from 80 to 125 %, which indicates the pharmacokinetic compared drugs equivalence. A total of 6 adverse events of mild and moderate severity were recorded. Of these, 4 adverse events were noted after administration of the study drug and were not associated with its administration. A comparative analysis of safety assessment parameters in this study (frequency and severity of adverse events, physical examination of healthy volunteers with an assessment of vital signs, laboratory tests, electrocardiography) did not reveal intergroup differences.Conclusion. The pharmacokinetic equivalence of the Glurazyme® and the reference drug in a dose of 30 U/kg has been established. A nonlinear dependence of the main pharmacokinetic parameters on studied drug administered dose was revealed. Safety and the absence of adverse reactions after a single injection of the study drug are shown.

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.

Представлены результаты исследований препарата Тигераза® (р-р для ингаляций, производитель АО «ГЕНЕРИУМ», Россия), проведённые для получения доказательств его подобности (сопоставимости) референтному препарату Пульмозим® (р-р для ингаляций, производитель «ХоффманнЛя Рош Лтд.», Швейцария). Оба препарата содержат в качестве действующего вещества человеческую рекомбинантную дезоксирибонуклеазу I — дорназу альфа, предназначены для лечения кистозного фиброза с лёгочными проявлениями (муковисцидоза). Изучена ферментативная активность дорназы альфа, содержащейся в изучаемых препаратах, in vitro и ex vivo на образцах гнойной мокроты больных. Изучены фармакокинетические параметры препаратов в сыворотке крови, бронхах и лёгких и основные физиологические показатели (масса и температура тела, состояние сердечно-сосудистой, дыхательной, выделительной систем, гематологические и биохимические параметры крови, патоморфологические изменения внутренних органов, включая состояние роговицы глаз, показатели смертности) в сравнительных исследованиях субхронической токсичности на ювенильных и половозрелых крысах при 28-дневном ингалировании в дозах 0,2 мг/кг половозрелым и 0,26 мг/кг ювенильным животным (доза в 6 раз превышала рекомендованную для клинического применения). Результаты исследований позволяют сделать вывод о сопоставимости препаратов по ферментативной, муколитической (секретолитической) ДНКазной активности, профилю безопасности и основным фармакокинетическим параметрам.

Background/Objectives: Risdiplam is a low-molecular-weight small-molecule modifier of SMN2 pre-mRNA splicing that was developed for spinal muscular atrophy (SMA) therapy and approved for the treatment of SMA as Evrysdi^® (Roche, Basel, Switzerland). Vapromin^® is a generic drug produced by JSC GENERIUM. In order to evaluate its biological activity and compare different batches of the reference drug and generic risdiplam, we performed comprehensive in vitro procedures. It is important to emphasize that this study did not assess the bioequivalence of the medicinal products for the purpose of comparing the biopharmaceutical quality of the generic and reference products. Instead, this study was designed specifically to compare their biological activity using cells derived from SMA patients and a reporter cell line. Methods: The biological activity of generic risdiplam was compared with that of the reference drug by assessing increases in SMN protein production and the relative transcription level of SMN2 mRNA in fibroblasts from SMA donors. Additionally, Exon 7 inclusion efficiency was evaluated using a constructed reporter cell line. Results: Using primary dermal fibroblasts from SMA probands, we demonstrated a concentration-dependent relationship between risdiplam concentration and SMN2 FL and SMN2 Δ7 transcript levels. At a risdiplam concentration of 0.18 μM, the relative SMN2 full-length transcript levels reached a maximum, with a mean 2.5-fold increase observed for both Evrysdi^® (Roche, Basel, Switzerland) and Vapromin^®. In primary fibroblasts derived from three SMA patients, the 2 SD quality range for Evrysdi^® (Roche, Basel, Switzerland) was 98.0–106.7% (σ(log RP) = 0.0092), and the activities of all Vapromin^® batches fell within this range. Using the reporter cell line, the quality range for Evrysdi^® (Roche, Basel, Switzerland) was 88.32–117.3% (σ = 0.0309), and the Vapromin^® values also fell within this range. Conclusions: This study experimentally confirmed that the in vitro biological activity of the generic drug Vapromin^® is comparable to that of Evrysdi^® (Roche, Basel, Switzerland). Further research should be conducted to confirm bioequivalence between the two products.

Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer. Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients. The development of monoclonal antibodies (mAbs) specific for PD-L1 or PD-1 is a promising area of immunotherapy of malignant tumours. However, before a therapeutic antibody-based product enters the market, it is necessary to ensure its safety and efficacy, i.e. perform a full scope of preclinical and clinical studies.The aim of the study was to develop and validate a bioanalytical method that does not require additional labeling and that could be used for determination of mAbs specific for human PD-L1 in the blood serum of a biological test system during preclinical studies.Materials and methods: an antigen in the form of a dimer of PD-1 extra-cellular domain covalently bonded to the Fc-fragment of human IgG (R&D Systems, USA) was used in the study. The antigen was immobilised on Dip and Read™ Protein A biosensors (Fortebio, USA). The therapeutic anti-PD-1 antibody GNR-051 was developed and produced by IBC “Generium” (Russia). The healthy cynomolgus monkey serum samples used as matrix were obtained from the Research Institute of Medical Primatology (Sochi, Russia). The assessment of binding was performed using Octet® QKe interferometer (Fortebio, USA) by real-time analysis of the dose-dependent rate of the antigen-antibody complex formation.Results: the paper presents experimental data on the development and validation of the test method for determination of the therapeutic PD-1-binding mAb concentration in cynomolgus monkey serum in the antibody concentration range from 2 to 2500 µg/mL. The authors assessed the calibration curve reliability, between-run and within-run precision and accuracy, dilution linearity, specificity and selectivity of the test method.Conclusions: the authors developed and validated the biolayer interferometry-based method for determination of therapeutic mAbs concentration. The method was shown to comply with the Eurasian Economic Union’s regulatory requirements in terms of the main validation parameters: analytical range, accuracy, precision, and selectivity.

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.