Rahim R. Shukurov

Fabry disease is a rare genetic multisystemic lysosomal disease characterized by progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs with specific cutaneous (angiokeratoma), neurological (pain), renal (proteinuria, chronic kidney failure), cardiovascular (cardiomyopathy, arrhythmia), cochleovestibular and cerebrovascular manifestations (transient ischemic attacks, strokes). The phenotypic expression depends on the age of onset and, in females, on the level of X-inactivation. Enzyme replacement therapy is currently the standard therapy for the disease. JSC GENERIUM recently developed a potential biosimilar of Fabrazyme^®, GNR-062, and investigated its physicochemical and biological properties, pharmacokinetics and immunogenicity to assess its bioequivalence to Fabrazyme^®. Direct evaluation of the N-glycosylation profile by HPLC revealed differences mainly related to the distribution of complex sialylated glycans. The overall sialylation level was comparable between the two products. The same conclusion follows from the results of direct analysis of the sialic acid content. Despite a small difference of a few percent in biphosphorylated glycan content, the overall measured M6P values were comparable. Thus, the internalization of agalsidase β into the inner space of lysosomes by binding to key receptors appear to be the same for both products. In a single-blind, randomized comparative study of the pharmacokinetics and safety of a single intravenous infusion of GNR-062 (JSC GENERIUM, Russia) and Fabrazyme^® (Genzyme Europe B.V., The Netherlands) in a crossover design in healthy volunteers, the ratio of geometric means (90% CI) of AUCinf and Cmax for GNR-062 to agalsidase β was 0.9736 (0.9413–1.0070) and 0.9867 (0.9546–1.0199), respectively. These studies demonstrated pharmacokinetic bioequivalence of GNR-062 to agalsidase β. Immunogenicity findings are preliminary, as the study was not powered for formal immunogenicity equivalence, and the single-dose design in healthy volunteers does not reflect repeated long-term administration in patients with Fabry disease. In conclusion, GNR-062 demonstrated acceptable short-term tolerability in healthy volunteers and warrants further investigation as a potential therapeutic option for patients with Fabry disease.

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.

GNR-068 is a proposed biosimilar to the ustekinumab reference product (RP), which works through the antagonism of interleukin 12 and interleukin 23. Ustekinumab RP is used for the treatment of chronic inflammatory conditions, including certain forms of plaque psoriasis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis. Objectives: The purpose of the study is to study the safety, pharmacokinetics, and immunogenicity of the ustekinumab biosimilar GNR-068 and the reference drug Stelara^® after a single subcutaneous administration in healthy male volunteers. Methods: This was an open-label, randomized, comparative, parallel-group clinical trial of the safety, pharmacokinetics (PK), and immunogenicity of GNR-068 (JSC GENERIUM) and Stelara^® (Manufacturer: Silag AG, Switzerland; RU Holder: JOHNSON & JOHNSON) after a single subcutaneous administration of 45 mg in healthy volunteers. During the trial, 146 volunteers were screened, and 122 were randomized. Results: The results showed that PK similarity was established based on 90% confidence intervals (CIs) for the ratios of geometric means of the primary endpoints of area under the concentration-time curve from time 0 extrapolated to infinity (AUC_0–∞) and maximum observed serum concentration (Cmax) being contained within the pre-specified margin of 80.00–125.00%. The incidence of total ADA was lower in the GNR-068 group compared with the reference product group. Adverse events were similar between treatment groups and consistent with the safety profile of the ustekinumab RP. Conclusions: These results indicate that GNR-068 and the ustekinumab RP share similar PK and safety profiles.

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.

Background/Objectives: Ocrelizumab is a humanized monoclonal antibody targeting CD20, approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). The neutralizing activity of anti-drug antibodies (ADAs), especially neutralizing ADAs (nADAs) activity, should be examined considering that it can alter pharmacokinetic (PK) and pharmacodynamic (PD) profiles, reduce drug efficacy, and lead to life-threatening adverse events. Methods: This article presents data on the development and validation of an assay for neutralizing anti-drug antibodies (nADA) based on ADCC reporter cells for the analysis of patient sera in the context of ocrelizumab clinical studies. Results: Critical steps and conditions to minimize assay variability were identified. The lower limit of detection was 549.6 ng/mL. The cutoff for nonspecific neutralization was determined as 19.7%. The presence of 0.37–3.0 μg/mL ocrelizumab in a biological sample enables the detection of 1.1–10.0 μg/mL polyclonal anti-ocrelizumab idiotype antibodies, respectively. Conclusions: The developed method can be used for immunogenicity studies of medicinal products containing ocrelizumab.

Background. Enzyme replacement drugs for treatment of mucopolysaccharidosis type II (MPS II) do not penetrate the blood-brain barrier, significantly reducing their efficacy in patients with neuropathic form. Verenafusp alfa (Clotilia®) is a recombinant fusion protein of iduronate-2-sulfatase and a monoclonal antibody Fab fragment to human insulin receptor for distribution of the enzyme into brain. Aim. To evaluate safety, tolerability, and pharmacokinetic parameters of verenafusp alfa after single intravenous administration of escalating doses in healthy volunteers. Materials and methods. This open-label, multicohort study included 20 healthy male volunteers aged 18 to 47 years (26.1±7.8 years). Verenafusp alfa was administered intravenously for 3 hours at single doses of 0.3 mg/kg (n=1), 0.5 mg/kg (n=1), 1 mg/kg (n=6), 2 mg/kg (n=6), and 3 mg/kg (n=6). Safety was assessed based on the incidence of clinical and laboratory adverse events (AEs) and their relationship to the investigational medicinal product. Pharmacokinetic parameters were calculated using a noncompartmental method. Results. All 20 volunteers completed the study. AEs reported in 6 (30%) volunteers were mild in severity and related to changes in individual laboratory and instrumental test data. One AE (increased bilirubin level) was possibly related to the study drug. Pharmacokinetic analysis demonstrated a dose-dependent increase in maximum concentration (Cmax) from 197.60 (0.3 mg/kg) to 10,225.80 ng/mL (3 mg/kg) and area under the concentration-time curve (AUC) from 38,678.60 to 2,714,067.42 ng×min/mL respectively. The half-life ranged from 86.69 to 213.42 minutes, and clearance lowered with the increasing dose from 7.67 to 1.11 mL/min/kg. Conclusion. Single intravenous administration of verenafusp alfa at doses ranging from 0.3 to 3 mg/kg demonstrated a favorable safety profile and good tolerability in healthy volunteers. The drug's pharmacokinetics was nonlinear, with a dose-dependent increase in Cmax and AUC with the dose increment. Volume of distribution volume lowered with increase of the dose.

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.

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.

SCIENTIFIC RELEVANCE. An important quality-control issue for therapeutic monoclonal antibodies (mAbs) is the determination of residual protein A leaching from the carrier during the purification of mAbs by affinity chromatography. However, unrelated compounds (immunoglobulins) present in the sample can complicate the immunoenzymatic detection of protein A (matrix effect), potentially leading to false-negative test results. To increase the efficiency of enzyme-linked immunosorbent assay (ELISA), it is necessary to develop a sample preparation step that can irreversibly break the bond in the protein A–mAb complex.AIM. This study aimed to develop and validate an analytical procedure for the determination of residual protein A in active substances of therapeutic mAbs by ELISA with a test kit comprising in-house reagents.MATERIALS AND METHODS. Recombinant protein A was used as an antigen. Polyclonal antibodies to protein A were produced by immunising chickens, selecting immunised eggs, and isolating antibodies from these eggs. Protein A-specific antibodies were purified by affinity chromatography. Residual protein A was determined using sandwich ELISA with preliminary sample preparation. The analytical procedure was validated in accordance with the requirements of the State Pharmacopoeia of the Russian Federation (Validation of Analytical Procedures, OFS.1.1.0012).RESULTS. The authors obtained, isolated, and purified chicken IgY antibodies to recombinant protein A. The authors selected sample preparation conditions for the determination of residual protein A by ELISA and optimum compositions of buffer solutions, including the composition of a denaturing buffer to disrupt the protein A–mAb complex. The developed analytical procedure was validated. According to the measurements of protein A, the accuracy of the analytical procedure ranged within 83–108% of the nominal value, the interlaboratory precision ranged within 96–116%, and the repeatability was up to 13%. The lower limit of quantitation was 10 ng/mL with a minimum required dilution of 1:10. The analytical range extended from 10 to 40 ng/mL. The analytical procedure showed results comparable to those obtained with a similar test kit from an international manufacturer.CONCLUSIONS. The developed analytical procedure for the determination of residual protein A by ELISA with a test kit comprising in-house reagents can minimise the matrix effect in therapeutic mAbs. This analytical procedure will alleviate import substitution and reduce quality control costs for Russian immunobiologicals.