This study aimed to determine the optimal cultivation conditions for four E. coli strains producing recombinant glucagon-like peptide-1 (GLP-1) tandems. Validation experiments confirmed that scale-up from shake flasks to a bioreactor is feasible without significant loss of specific productivity. We also demonstrated that the optimal cultivation conditions are identical for all tested strains. However, the number of GLP-1 monomers in the tandem influenced the final yield in both shake flasks and the bioreactor. The quality of the resulting inclusion bodies (IBs) was also evaluated. The IB content per gram of biomass was found to correlate with specific productivity, and IB quality varied among the different tandems. A correlation was identified between the titer of the GLP-1 tandems and the number of monomers. The results indicate that selecting the appropriate number of monomers during strain development can enhance productivity, improve inclusion body quality, and increase the final product titer after downstream processing.

Ultra-rapid insulin lispro is an innovative insulin analogue designed to achieve rapid onset and short duration of action, aimed at optimizing glycemic control in patients with diabetes. This was a double-blind, randomized, 2-period, crossover clamp study to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD), along with safety profiles, of a potential biosimilar ultra-rapid insulin lispro compared to the reference product in healthy White men. A total of 35 healthy volunteers completed hyperinsulinemic euglycemic clamp procedures across both study periods. Blood samples were collected at predefined intervals up to 8 hours to assess PK parameters. Plasma glucose levels were monitored every 5 minutes during the 8-hour clamps, with adjustments to the glucose infusion rate to maintain the target range. Insulin quantification in plasma was conducted using a validated enzyme-linked immunosorbent assay method. PD assessment was based on glucose infusion rate profiles during both clamps. Geometric mean ratios for maximum plasma concentration and area under the concentration-time curve from insulin administration to the last measurable concentration for the test and reference drugs fell within the bioequivalence range of 80%-125%. Furthermore, the investigational drugs demonstrated comparable PK/PD profiles of insulin lispro. Both formulations exhibited similar safety profiles primarily characterized by mild injection site reactions.

AIMS:To evaluate the immunogenicity, efficacy, and safety of a biosimilar insulin glulisine candidate (T-Glu) compared to the reference product (R-Glu) in patients with type 1 diabetes mellitus (T1DM). MATERIALS AND METHODS:In this phase III, randomised, open-label trial, adult patients with T1DM received either T-Glu or R-Glu by prefilled pens as their bolus insulin for 26 weeks. The primary outcome was the proportion of patients with an immune response at Week 26. Secondary endpoints included changes in glycaemia control parameters, insulin dose stability, treatment satisfaction, and other immunogenicity and safety outcomes. RESULTS:By Week 26, the immune response persisted in 16% of patients in the T-Glu group and 24% in the R-Glu group (p > 0.05), confirming similar immunogenicity. HbA1c reduction at Week 26 compared to baseline was similar between groups, with a mean change of 0.53% ± 1.19% in the T-Glu group and 0.38% ± 1.46% in the R-Glu group (mean difference: -0.15%, 95% CI: -0.50 to 0.20), demonstrating non-inferiority (according to a predefined margin of 0.4%). Fasting plasma glucose levels and the seven-point glucose profile showed no significant differences between groups (p > 0.05). Insulin dose remained stable throughout the study, and treatment satisfaction scores improved comparably in both groups (p > 0.05). The total number of AEs was similar, and hypoglycaemia was more frequent in the R-Glu group (p < 0.001), with no new safety concerns identified. CONCLUSIONS:T-Glu demonstrated similar immunogenicity, efficacy, and safety to R-Glu in adults with T1DM, supporting its use as a biosimilar insulin glulisine. CLINICALTRIALS:gov: NCT07070752.

GP40141 is a romiplostim biosimilar. A Phase 1 clinical trial was previously conducted in healthy volunteers to evaluate the pharmacodynamics (PD), pharmacokinetics (PK), and safety of GP40141 compared to the reference romiplostim (NCT05652595). Using noncompartmental analysis, the biosimilarity of PD end points was determined according to the classical criterion (0.8-1.25). PK end points were also in good agreement between GP40141 and the reference romiplostim; however, the confidence interval for the area under concentration-time curve from time 0 to the time of last measurement was slightly out of the bioequivalence range (0.91-1.29). Population PK/PD was used in the present study to characterize the individual PK and PD data of 56 healthy subjects in 2 cross-over periods of the Phase 1 clinical trial. Body weight and neutralizing antibodies to romiplostim were found to be important predictors of apparent volume of distribution and linear elimination constant, respectively. Within the framework of the conducted modeling, population estimates of PK/PD parameters were obtained, which were in agreement with literature data for the reference romiplostim. Additionally, values of intersubject variability, previously unreported for romiplostim in a healthy subject population, were derived. Covariate analysis, conducted during model development, as well as visual diagnostics and model-based simulations, demonstrated the absence of significant differences in PK and PD between GP40141 and romiplostim-ref.

During biosimilar drug development, conducting a clinical trial of biosimilar efficacy in patients may become necessary in the presence of residual uncertainty regarding the biosimilarity of the drugs. In the development of the biosimilar romiplostim GP40141, we aimed to use a model-based in silico clinical trial (ISCT) approach to optimize the planned biosimilar efficacy trial in patients with immune thrombocytopenia. The population pharmacokinetic/pharmacodynamic model for healthy volunteers was modified and validated to describe platelet dynamics in patients with immune thrombocytopenia. ISCTs were then conducted using the modified model for various expected scenarios of biosimilar efficacy trials. Statistical analysis of the simulation results was subsequently used to confirm the appropriateness of the chosen design for evaluating the planned efficacy end points. Since the planned trial includes both patients naïve to therapy with thrombopoietin receptor agonists and nonnaïve patients, various expected ratios of naïve to nonnaïve patients (1:1, 1:2, 1:3) and the percentage of nonnaïve patients who previously received eltrombopag (0% or 30%) were assessed across 200 ISCTs performed for each scenario. The obtained estimates of empirical power for the equivalence test of platelet response/durable platelet response by the 10th/26th week between the test and reference groups were not less than 94%, regardless of the scenario. Differences in power between the 10- and 26-week end points did not exceed 4%. The analysis of ISCT results allowed for an effective reduction of uncertainty in the biosimilar development of GP40141, demonstrating the appropriateness of using the 10-week efficacy end point as the primary one.

The aim of the study was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of T-glu (GP40321, test drug), and reference insulin glulisine in a hyperinsulinemic-euglycemic clamp procedure. During this study, 34 healthy male volunteers underwent the hyperinsulinemic-euglycemic clamp procedure following subcutaneous 0.3 U/kg injection of T-glu or reference insulin glulisine in a randomized, double-blind, crossover study. Plasma glucose levels were monitored every 5 minutes for 8 hours. Glucose infusion rate adjustment was based on the blood glucose measurements. Evaluation of PD was performed using the glucose infusion rate values, while PK was calculated using insulin concentrations measured via enzyme-linked immunosorbent assay. The study results showed that the 90% CI for the geometric mean ratios of primary PK and PD of T-glu and reference insulin glulisine were within 80%-125% comparability limits, and that the safety profiles were comparable. PK, PD, and safety similarity of T-glu and reference insulin glulisine was demonstrated.

This study was conducted as a single-site, open-label, randomized, replicated crossover trial with 4 treatment periods. The aim was to evaluate the bioequivalence of a generic test drug containing velpatasvir and sofosbuvir compared to an established brand-name medication in healthy White subjects under fasting conditions. Blood samples were collected at specified intervals up to 72 hours after dosing to measure the concentrations of velpatasvir and sofosbuvir using a certified high-performance liquid chromatography with tandem mass spectrometry method. The bioequivalence of the 2 formulations was confirmed when statistical analysis showed that confidence intervals for the log-transformed peak concentration and area under the concentration-time curve from time 0 to the last quantifiable sample were within an acceptable range from 80% to 125%. Criteria for bioequivalence were met for both area under the concentration-time curve from time 0 until the last quantifiable sample and peak concentration parameters. No adverse effects were reported during this trial in both groups.

GP40141 is a romiplostim biosimilar. A Phase 1 clinical trial was previously conducted in healthy volunteers to evaluate the pharmacodynamics (PD), pharmacokinetics (PK), and safety of GP40141 compared to the reference romiplostim (NCT05652595). Using noncompartmental analysis, the biosimilarity of PD end points was determined according to the classical criterion (0.8-1.25). PK end points were also in good agreement between GP40141 and the reference romiplostim; however, the confidence interval for the area under concentration-time curve from time 0 to the time of last measurement was slightly out of the bioequivalence range (0.91-1.29). Population PK/PD was used in the present study to characterize the individual PK and PD data of 56 healthy subjects in 2 cross-over periods of the Phase 1 clinical trial. Body weight and neutralizing antibodies to romiplostim were found to be important predictors of apparent volume of distribution and linear elimination constant, respectively. Within the framework of the conducted modeling, population estimates of PK/PD parameters were obtained, which were in agreement with literature data for the reference romiplostim. Additionally, values of intersubject variability, previously unreported for romiplostim in a healthy subject population, were derived. Covariate analysis, conducted during model development, as well as visual diagnostics and model-based simulations, demonstrated the absence of significant differences in PK and PD between GP40141 and romiplostim-ref.

Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies.

The progressive nature of type 2 diabetes mellitus leads to the need for insulin therapy in a significant proportion of patients. Very often start of insulin therapy in type 2 diabetes mellitus (T2DM) is associated with weight gain and a significant increase of hypoglycemia’s risk. However, innovative options, such as fixed ratio combinations of glucagon-like peptide 1 receptor agonists (GLP-1RA) and basal insulin, minimize weight gain and hypoglycemia risks and allow a greater proportion of patients to achieve individual glycemic control goals without compromising safety parameters. This review includes a description of the randomized clinical trials, as well as the results of real clinical practice of the use of two currently existing fixed ration combinations of GLP-1RA and basal insulin — iDegLira and iGlarLixi.

The aim of the study was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of T-glu (GP40321, test drug), and reference insulin glulisine in a hyperinsulinemic-euglycemic clamp procedure. During this study, 34 healthy male volunteers underwent the hyperinsulinemic-euglycemic clamp procedure following subcutaneous 0.3 U/kg injection of T-glu or reference insulin glulisine in a randomized, double-blind, crossover study. Plasma glucose levels were monitored every 5 minutes for 8 hours. Glucose infusion rate adjustment was based on the blood glucose measurements. Evaluation of PD was performed using the glucose infusion rate values, while PK was calculated using insulin concentrations measured via enzyme-linked immunosorbent assay. The study results showed that the 90% CI for the geometric mean ratios of primary PK and PD of T-glu and reference insulin glulisine were within 80%-125% comparability limits, and that the safety profiles were comparable. PK, PD, and safety similarity of T-glu and reference insulin glulisine was demonstrated.

2021 marks the 100th anniversary of the discovery of insulin, an event that forever changed the lives of people with diabetes mellitus. At present patients around the world experience the miracle of insulin therapy every day. A disease that used to kill children and teenagers in 2 years in 1920 has become a disease that can be controlled with a possibility to lead a long productive life. Over the past century, the great discovery of Banting, Best and Collip has forever changed the world and saved millions of lives. This review is devoted to the history of the development of insulin and its further improvement: from the moment of discovery to the present days. Various generations of insulin are considered: from animals to modern ultrashort and basal analogues. The article ends with a brief review of current trends in the development of new delivery methods and the development of new insulin molecules. Over the past century, insulin therapy has come a long way, which has significantly improved the quality of life of our patients. But research is actively continuing, including in the field of alternative methods of insulin delivery, which are more convenient for the patient, as well as in the development of «smart» molecules that will have a glucose-dependent effect.

Introduction. Over the years, insulin therapy has remained an important component of the complex treatment of patients with diabetes mellitus. Ultra-rapid insulin lispro is a DNA recombinant analogue of human insulin, which has a pharmacokinetic (PK) profile that is as close as possible to the endogenous insulin secretion profile, which ensures effective control of postprandial glycaemia. The development of the ultra-rapid insulin lispro biosimilar will expand the range and increase the availability of modern and safe insulin analogues for diabetic patients in Russia.Aim. To compare the PK and pharmacodynamic (PD) profiles of GP40261 (ultra-rapid insulin lispro biosimilar) and the reference Lyumjev® in healthy volunteers.Materials and methods. This was a double-blind, randomised, comparative, crossover study in healthy volunteers who were administered either the test or reference ultra-rapid insulin lispro formulation as a single dose of 0.3 IU/kg. The hyperinsulinaemic euglycaemic clamp technique was used to evaluate the pharmacokinetics and pharmacodynamics of the study products. In order to assess the biosimilarity of the products, 90% confidence intervals (CIs) were calculated for geometric mean ratios of the primary PK parameters AUCins.0-t and Cins.max. The PD parameters of the study drugs were evaluated based on the glucose infusion rate required to maintain the target glycaemic level during the clamp.Results. The 90% CIs for the geometric mean ratios of the primary PK parameters for the test and reference products were 89.41-94.55% for AUCins.0-t and 82.74-92.92% for Cins.max, which complies with the established acceptance limits of 80-125% for both parameters. The study products were also found to have comparable PD profiles of their active substances.Conclusion. This clinical study has demonstrated that GP40261 and the reference ultra-rapid insulin lispro are biosimilar and have a comparable safety profile.

This study was conducted as a single-site, open-label, randomized, replicated crossover trial with 4 treatment periods. The aim was to evaluate the bioequivalence of a generic test drug containing velpatasvir and sofosbuvir compared to an established brand-name medication in healthy White subjects under fasting conditions. Blood samples were collected at specified intervals up to 72 hours after dosing to measure the concentrations of velpatasvir and sofosbuvir using a certified high-performance liquid chromatography with tandem mass spectrometry method. The bioequivalence of the 2 formulations was confirmed when statistical analysis showed that confidence intervals for the log-transformed peak concentration and area under the concentration-time curve from time 0 to the last quantifiable sample were within an acceptable range from 80% to 125%. Criteria for bioequivalence were met for both area under the concentration-time curve from time 0 until the last quantifiable sample and peak concentration parameters. No adverse effects were reported during this trial in both groups.

Introduction. The production of Fc-fused proteins in prokaryotic systems often results in the formation of insoluble aggregates due to improper folding of polypeptide chains. To obtain functional proteins, a refolding step is required. However, developing refolding parameters can be time-consuming. The optimization of renaturation conditions using the Design of Experiments (DoE) approach allows for the calculation of optimal process parameters and the evaluation of contributing factors and their interactions.Aim. This study aims to evaluate the effects of denaturation buffer pH, as well as oxidative and reducing agent concentrations, on the efficiency of Fc-fusion protein refolding in vitro and to determine optimal refolding parameters.Materials and methods. Fc-fusion protein inclusion bodies were obtained from an Escherichia coli BL21 bacterial expression system. The experiment was designed using an orthogonal composite design (Orthogonal Central Composite Design, CCO). Experimental design, statistical data processing, and parameter optimization were conducted using MODDE (v. 12.1, Sartorius Stedim Data Analytics AB, Germany). Chromatographic purity and yield of the target protein, as determined by high-performance size-exclusion chromatography, were used as response variables.Results and discussion. The DoE approach successfully optimized the Fc-fusion protein refolding process. Response surface plots were constructed, and the optimal factor values were determined. The statistical models demonstrated high predictive accuracy and data reproducibility. The refolding process was successfully validated under optimized conditions, resulting in a decrease in high-molecular-weight impurities and improperly folded protein forms. The chromatographic purity of the target protein increased by more than 10 %, as confirmed by high-performance size-exclusion chromatography.Conclusion. The study established significant effects of buffer pH, redox pair concentrations, and their interactions on the yield and chromatographic purity of the Fc-fused protein. The interplay between oxidative and reducing components and buffer pH was demonstrated. Increasing the buffer pH led to improved refolding efficiency.

The aim of the work is to conduct a review of medications approved by the FDA in 2022. Materials and methods. In searching for the materials to write this review article, bibliographic databases including PubMed, Google Scholar and e-library.ru were utilized. The search was conducted for the publications spanning the period from 2008 to 2023. Herewith, the following keywords and word combinations were used: new drug approval, NDA, drug authorization, approval package, breakthrough medicine. Results. The discovery and development of medications are among the most crucial scientific processes in healthcare. Developing a new drug is a highly intricate, expensive, and time-consuming process. Nowadays, the problem of costs reduction and the process of expedited discovering of new medications are particularly pertinent. To optimize the search for active compounds, virtual and high-throughput screenings, machine learning, artificial intelligence, cryo-electron microscopy, and drug repurposing are employed. Simultaneously, the search for original molecules to serve as the basis for innovative drugs continues. This article presents a review of medications approved by the FDA in 2022 for the treatment of various pathologies. Conclusion. A drug development is a complex and resource-intensive process, with only a small fraction of candidates advancing to clinical trials. A drug design evolves in tandem with societal needs, and this review highlights some of the medications approved by the FDA in 2022. Technological advancements are expected to expedite drug development, potentially reducing the time to the market. Biotechnology, including cell therapy, holds significant prospects, and achievements in genetic mapping and chip technologies will enhance the accessibility of personalized pharmacology.

Abstract Aims The pharmacodynamic (PD) similarity between GP40141, a proposed romiplostim biosimilar, and reference romiplostim was evaluated. Pharmacokinetics and safety were also assessed. Methods In this phase 1, randomized, double‐blind, single‐dose, crossover comparative study with an adaptive design, 56 healthy male volunteers were randomized 1:1 to receive a 3 ug × kg−1 subcutaneous dose of GP40141 and reference romiplostim. The PD similarity between GP40141 and the reference romiplostim was determined using the standard equivalence criteria (80%–125%) for the area under the platelet count‐time curve from time 0 to the time of the last sampling for PD (AUCplt) and the maximum observed platelet count (Pmax). Results GP40141 and the reference romiplostim exhibited similar PD profiles. 90% CI for the geometric mean ratios for the primary PD parameters (AUCplt, Pmax) for GP40141 (T) and the reference romiplostim (R) were fully contained within the predefined equivalence limits of 80%–125%: 98.13%–102.42% for AUCplt and 97.56%–105.80% for Pmax. The pharmacokinetic profiles of GP40141 and the reference romiplostim were well described. No adverse events were observed during the clinical trial after the administration of GP40141 and the reference romiplostim. Conclusion This study demonstrates the PD similarity of GP40141 to the reference romiplostim. Both treatments had comparable safety profiles (NCT05652595)

Diabetes poses a serious threat to the health of people around the world. Therefore, in 2021, the World Health Organisation launched the Global Diabetes Compact, an initiative aimed at improving the management and prevention of diabetes. The rapid growth in the number of diabetic patients has increased the need for insulin. Rapid-acting human insulin analogues, including insulin aspart, improve the efficacy of insulin therapy. Methods for insulin aspart production include its biosynthesis in the proinsulin form in Escherichia coli . However, the yield of the recombinant protein largely depends on the optimisation of the production process. The aim of the study was to optimise the induction conditions for an E. coli strain expressing recombinant proinsulin aspart through applying the Design of Experiment (DoE) approach to enhance bacterial cell productivity. Materials and methods. The study focused on a strain of E. coli producing proinsulin aspart. The authors planned the experiment using MODDE software and the reduced face-centred central composite design (CCF) enabling the assessment of factor interactions and the creation of design spaces. The authors carried out fermentations of the producing strain in a 5 L Biostat® B bioreactor and measured proinsulin aspart concentrations by capillary gel electrophoresis. The results were analysed using GraphPad Prism 6. Results. Using the DoE approach, the authors optimised the conditions for the growth of the producer strain and the biosynthesis of proinsulin aspart. Based on data from response surface plots for wet biomass concentration, specific productivity, and volumetric productivity, as well as plotted models, the authors established design spaces for the induction of proinsulin aspart expression in E. coli . The plotted models demonstrated high predictive power and high reproducibility of the results. The authors successfully validated the induction process for the synthesis of proinsulin aspart in a bioreactor under optimised conditions. The volumetric productivity of the strain producing proinsulin aspart increased from 3.06±0.16 g/L (conventional conditions) to 4.93±0.80 g/L (optimised conditions). Conclusions. The authors achieved a 60% increase in the volumetric yield of proinsulin aspart. The study results may be used to intensify the industrial production of insulin aspart.

The aim of the work was to conduct an analysis of the current state and current trends in the approval of drugs, as well as some aspects of the methodology for their development based on biological molecules and registration. Materials and methods. The material for the analysis was taken from the abstract databases of PubMed, Google Scholar and e-library.ru. The search was carried out using publications for the period from 2008 to 2023, the keywords were as follows: "biologicals", "new drug approval", "drug authorization", "drug development", "biosimilar", taking into account various spellings. Results. Over the past 15 years, scientists have been observing revolutionary trends and processes in the field of the drug development, especially biopharmaceuticals. Significant advances have been made in gene, immune and cell therapies, resulting in the approval of such drugs more than doubling over the past ten years. The development of biological drugs includes the identification and testing of molecular targets and requires a deep understanding of the structure and functioning of the polypeptides involved in the development of the effect. The features of these active pharmaceutical substances are a high molecular weight, a complex three-dimensional structure and a high immunogenic potential. Preclinical and clinical studies of biologics have unique challenges. Selecting appropriate animal species, understanding the immunogenicity, and assessing pharmacodynamics and toxicological properties require a multilevel, detailed approach. The article discusses the regulatory framework under which these drugs are registered, summarizing the guidelines provided by international organizations such as the International Council for Harmonization and various national agencies. Conclusion. The analysis highlights the current advances and prospects in the development of biologics, highlighting their key role in future transformations in the treatment of rare diseases and oncology, approaching the era of personalized medicine. Monitoring the development directions and technological approaches, as well as the commitment to global methodological and regulatory aspects can become a catalyst in the development of the Russian pharmacology.