A study of the degradation profiles of drugs based on recombinant monoclonal antibodies under stress conditions made it possible to determine the dynamics and mechanisms of degradation processes, to identify drug degradation products, and to reveal differences in the stability of biosimilar drugs for relatively short times. A strategy for accelerated (35 d) stability assessment under stress conditions of the original product Soliris® (Switzerland) and its biosimilar PRK-001 (Russia) was proposed. Their degradation profiles after exposure to extreme pH, oxidative stress, and UV radiation were studied. Antibody fragments were analyzed using size-exclusion chromatography, capillary isoelectric focusing, electrophoresis, and mass spectrometry. Both products showed similar degradation profiles and similar changes in isoform contents.

Исследование профилей деградации лекарственных препаратов на основе рекомбинантных моноклональных антител в стрессовых условиях позволяет определить динамику и механизмы процессов деградации, идентифицировать продукты распада препарата, выявить различия в стабильности биоподобных препаратов за относительно короткое время. В работе предложена стратегия ускоренной (35 сут) оценки стабильности в стрессовых условиях оригинального продукта Soliris® (Швейцария) и его биоаналога ПРК-001 (Россия), проведено исследование их профилей деградации в условиях экстремальных рН, окислительного стресса и под действием УФ-излучения. Фрагменты антител анализировали с помощью гельпроникающей хроматографии, капиллярного изоэлектрического фокусирования, электрофореза и масс-спектрометрии. Оба продукта продемонстрировали близкие профили деградации и аналогичные изменения в содержании изоформ.

Soliris® (Eculizumab) was approved by regulatory authorities as the first drag for treatment of orphan disease, paroxysmal nocturnal hemoglobinuria, in 2007. Later its use was extended for treatment of atypical hemolytic-uremic syndrome and myasthenia gravis. The high cost, the unavailability of therapy for a number of patients, as well as the expiration of the patent protection period for Soliris®, these factors became the prerequisite for the development of biosimilar medicinal products. Here, comparative analysis of PRK-001 (LLC «Pharmapark») and reference drag product, Soliris® (Alexion Pharmaceuticals, USA), was carried out. Physicochemical biosimilarity assessment has shown complete comparability of both products in terms of protein sequence as well as higher order structures. Post-translation modifications as well as impurity profile were the same too. Neither biosimilar nor reference product had impurity amount exceeding threshold. Biological properties of the reference product and biosimilar were the same. Since the comparability of the both drags has been proven, it can be assumed that PRK-001 is biosimilar of Soliris®. Eculizumab, Soliris, paroxysmal nocturnal hemoglobinuria, physicochemical comparison, post-translational modifications, biosimilar, drag product

Introduction. The drugs affecting a mitochondrial dysfunction, oxidative stresses, apoptosis and inflammation of the vascular wall, have a high potential for the prevention and treatment of atherosclerotic lesions. In this regard, the use of EPOR/CD131 heteroreceptor agonists which have a similar spectrum of pharmacological effects, is one of the promising strategies in the treatment of cardiovascular diseases.Materials and Methods. The study was carried out on 68 C57Bl/6J male mice. Atherosclerosis was simulated in transgenic animals with an endotheliospecific knockdown of the Polg gene by simulating a balloon injury and keeping on a Western diet. Then, the studied drugs were injected once every 3 days at the dose of 20 μg/kg for 27 days. On the 28-th day, the animals were euthanized and the area of atherosclerotic plaques was assessed. The gene expression associated with the processes of inflammation, antioxidant protection, apoptosis, and angiogenesis was also determined in the aortic tissues. In addition, the endothelium protective effect of peptides on primary cultures of endothelial cells of wild and transgenic Polg-D257A mice was studied.Results. No statistically significant effect of drugs on the area of lipid infiltration have been found. However, the studied peptides have significantly reduced the expression of proinflammatory genes (iNos, Icam1, Vcam1, Sele, Il6, Tnfa), the genes associated with angiogenesis (Vegfa, Kdr, and Hif1a), the expression of proapoptic factors; they decreased the Bax/Bcl-2 ratio by more than 1.5 times. In addition, when supplemented with H2 O2 in vitro, peptides dose-dependently increased endothelial cell survival.Conclusion. The erythropoietin-based peptides can be used to improve the functional state of the vascular wall against the background of atherosclerotic lesions and have a depressing effect on pathobiological processes associated with a mitochondrial dysfunction. In addition, the studied peptides have a significant endothelial protective effect in the induction of oxidative stress in vitro.

The correction of free radical oxidation processes is one of the most promising strategies of neuroprotection in acute cerebrovascular disorders. The aim of the study is an experimental study of the neuroprotective effects of 3-hydroxypyridine and erythropoietin derivatives, as well as their combined use. Materials and methods . The study was performed on 109 male Wistar rats. The neuroprotective effect of the substances was studied on a hemorrhagic stroke model. The study drugs were administered to the animals intraperitoneally. Carbamylated darbepoetin was administered three times in advance at the dose of 100 µg/kg within intervals of 3 days, the last injection took place 1 hour before the operation (the total dose was 300 mg/kg). Etoxidol was administered once 1 hour before the surgery at the dose of 50 mg/kg. The survival rate, behavioral features and the state of the animals on the 1st, 3rd, 7th and 14th days were recorded, and the morphological assessment of the brain was carried out. Results . The investigated substances had a positive effect on both the survival rate of the animals during the first day and on the 14th day. The best survival rates on the 14th day were recorded in the group of a combined use of ethoxydol and carbamylated darbepoetin (75%). Thus, in this group of rats, a faster recovery of neurological disorders was already distinguished from the first day on. By the 7th day, more than 50% of the rats receiving the combination of the studied drugs, had had a slight neurological deficit (up to 3 points on the McGrow scale); by the 14th day there had been only minor changes in the neurological status in the rats of this group. A pronounced neuroprotective effect of the combination of 3-hydroxypyridine and erythropoietin derivatives has been confirmed by a histological examination of brain slices – a more rapid decrease in the size of perifocal edema and microcirculation disorders, less damage to neurons and glial elements, and faster processes of resorption and organization of hemorrhage. A macroscopic examination of the brain sections stained with triphenyltetrazolium chloride of the dying rats, showed that perifocal necrosis had been the main cause of high mortality in the control group after the 3rd day. Conclusion . As a result of the experiment, the nephroprotective effect of the studied derivatives of 3-hydroxypyridine and erythropoietin has been proved. Moreover, the combination of these drugs has shown a greater neuroprotective activity than their isolated use. The additive effect of these drugs was due to their action mechanism resulting from the synergism of various structures and components of the cells.

AIM:to determine the efficiency of different doses of erythropoietin and carbamylated darbepoetin, to prove experimentally the possibility of increasing the efficiency of carbamylated darbepoetin when using the levorotatory stereoisomer of ethoxidol.MATERIALS AND METHODS:On the model of ischemia-reperfusion injury in male CD-1 mice that were undergone to contralateral nephrectomy, the nephroprotective effect of different doses of erythropoietin alfa, carbamylated darbepoetin, L-ethoxidol (the levorotatory enantiomer of ethylmethylhydroxypyridine malate), and the combined use of L-ethapylated carboxypyridine malate and L-etapoietin alfa was studied. The parameters of microcirculation and glomerular filtration rate were studied one day after 30-minute ischemia.RESULTS:It has been established that the prophylactic use of erythropoietin alfa and carbamylated darbepoetin in the model of ischemia-reperfusion injury of a single kidney reduces the severity of microcirculatory impairment and ensures the preservation of the glomerular filtration rate in dose-dependent manner. The synergistic effect of L-ethoxidol and carbamylated darbepoetin was found when these substances were used together.CONCLUSION:Prevention of ischemia-reperfusion kidney injury by analogs of human erythropoietin and their combination with ethylmethylhydroxypyridine derivatives is experimentally-proved.

Activation of the cerebral erythropoietin system can be a promising strategy for the management of various neurodegenerative and neuropsychiatric diseases as it triggers neuroprotective mechanisms and improves cognitive functions. Lack of information about the possible neurotrophic effect of erythropoietin reduces the possibility of using the brain receptor for erythropoietin as a therapeutic target in neurodegenerative diseases associated with hypoxia and inflammation. The investigation aims to study the influence of the erythropoietin receptor agonist - carbamylated darbepoetin (CdEpo) - on the morphofunctional features of neuron-glia networks of primary hippocampal cultures in mice under normoxia. Materials and Methods. Primary hippocampal cell cultures dissociated from embryos (E18) of C57BL/6 mice were used to study the influence of erythropoietin receptor stimulation on the functional activity of hippocampal neuron-glia networks. The experiments were carried out on days 18-23 of culture development in vitro. CdEpo (100 ng/ml) action duration was 24 h. Functional changes were assessed based on the electrical and metabolic activity of cultured cells using patch-clamp techniques, multielectrode registration of bioelectric activity in neural networks and calcium imaging, respectively. The morphological features of primary hippocampal cell cultures were studied using transmission electron microscopy. Results. Treatment of primary hippocampal cell cultures with CdEpo during 24 h did not affect the frequency of spontaneous single action potentials, spontaneous burst activity of cells, the patterns of neuron action potentials (the amplitude of depolarization phase, threshold potential, the amplitude of hyperpolarization phase), membrane capacity. It did not affect the bioelectric parameters of neural network activity (the number of spikes in a network burst, network burst duration, and inter-burst interval), calcium activity of neurons and glial cells determined by the duration and frequency parameters of spontaneous calcium oscillations. At the ultrastructural level, the number of mature asymmetric synaptic contacts remained unchanged under the influence of CdEpo, but there was a morphogenesis of the internal structure of dendritic spines: the number of spines with endoplasmic reticulum and/or the spine apparatus inside increased, which was a unique phenomenon for a model of primary hippocampal culture. Conclusion. There was revealed no neurotropic effect of CdEpo on the model of primary hippocampal culture as reflected by bioelectric activity parameters of single neurons, neuronal networks, and astrocytic network activity evident in the parameters of intracellular calcium concentration changes (calcium oscillations) under normoxia conditions. However, CdEpo causes changes in the internal structure of dendritic spines in some neurons with the spine apparatus appearing in them. The absence of CdEpo effect on the functions of intact neurons and glia indicates the relative safety of using this molecule for therapeutic purposes as cytoprotection for the brain tissue.

Method of determination of potassium cyanate in carbamylated darbepoietin alpha drug substance was developed. The method is based on derivatization of cyanate by methyl ester of anthranilic acid followed by formation bicyclic derivative (1-H,3-H-quinazolinedione), which is able to fluorescence. Application of this method to determine potassium cyanate in protein drugs was investigated. Interference of excipients on this method was evaluated. The validation was performed: specificity, linearity, accuracy, precision, limit of quantitication.

Two licensed vaccines Gardasil and Cervarix, containing main capsid protein L1 virus-like particles antigen, are used for prevention of diseases associated with human papilloma virus. L1 cannot provide protection against all pathogenic strains of HPV, shows low efficacy in middle and old age vaccinated patients, and are not therapeutically effective. The next generation of vaccines that contain minor protein L2 conserved epitopes as well as oncoproteins E6 and E7 promise to provide broad protection against all HPV, and prevent the emergence and spread of skin lesions.

The aim of the study was to test whether P-αB can be positioned as a preventing and treating agent for cardiovascular diseases.Materials and methods. The study was performed on sexually mature male Wistar rats. Endothelial dysfunction was modulated by a 7-days intraperitoneal administration of L-NAME at the dose of 2.5 mg/100 g. P-αB, or erythropoietin (EPO), was used for therapy at the dose of 2.5 µg/100 g × 3 times for 7 days, the total dose was 7.5 µg/100 g. The function of endothelium was estimated by an endothelium-dependent and endothelium-independent vasodilation. In addition, a histological assessment of the abdominal aortic wall state and the analysis of eNos, Tnf and Il-1β genes expression were performed. To estimate prothrombotic properties, P-αB and EPO were administered, at the doses of 2.5 and 5 µg/100 g (3 times a day for 7 days, the total doses were 7.5 µg/100 g and 15 µg/100 g, respectively) and on the 8th day, the time of ferric (III) chloride-induced carotid artery thrombosis was estimated.Results. Theresults of the functional tests for endothelium-dependent and endothelium-independent vasodilatation, as well as the histological picture of the aorta have evidenced that P-αB and EPO do not affect L-NAME-induced hypertension but improve the endothelium function. At the same time, P-αB shows a significantly higher endothelial-protective activity, reducing the coefficient of endothelial dysfunction from 5.1±0.15 to 2.72±0.12. In addition, P-αB has significantly increased the expression of eNos and reduced the expression level of Tnf and Il-1β mRNA genes. Carrying out Ferric (III) chloride-induced carotid artery thrombosis has revealed that P-αB (5 µg/100 g × 3 times a day for 7 days, total dose was 15 µg/100 g) has a lower but statistically significant prothrombotic activity than EPO.Conclusion. P-αB can be positioned as an atheroprotector because of its ability to prevent the death of endothelial cells, as well as to reduce remodeling and proinflammatory activation of the vascular wall. However, the prothrombotic properties of P-αB limit its use as a preventing and treating agent for atherosclerosis-associated diseases.

Ischemic stroke is one of the most frequent causes of death and disability of patients. Microglia is a unique example of immunocompetent cell in central nervous system that is capable protecting the brain from ischemic injury. Microglia are constantly surveying their microenvironment. Under pathological conditions microglia rapidly change their morphology and adopt activation states in order adequately react to the activation-causing stimuli. However, now there is no understanding of the specific physiological role of each of the morphological microglia phenotypes [1, 3, 6]. At present, drugs mainly aimed at minimizing the consequences of a stroke are intensely developed. One of the new lines in the development of neuroprotection methods in a stroke is to use a βcommon receptor subunit (β-cR) with EPOR as a therapeutic target. One of microglia membrane’s receptors is common receptor subunit (β-cR), which in the presence of a ligand can form a dimeric complex with erythropoietin. Carbamylated darbopoietin (CdEPO) (PHARMAPARK LLC, Moscow) was used as an agonist for this receptor. When the receptor is connected to erythropoietin, this triggers two types of reactions, namely, a positive one, which activates the protective mechanisms of damaged neurons, and a negative one, which is caused by the increasing number of erythrocytes, resulting in reformation of a thrombus and a rise in blood pressure [2,4]. Therefore, Carbamylated darbopoietin (CdEPO) (Pharmapark LLC, Moscow) is able protect neurons from ischemic damage, ignoring hemopoiesis, which prevents the increase in hematocrit. Approximately 80% of the thrombotic or thromboembolic strokes occur in the middle cerebral artery. Thus, a model of transient occlusion of the middle cerebral artery (tOCMA) was used to study the stroke pathogenesis mechanism [5]. The somatosensory cortex and the hippocampus play an important role in learning and memory processes. Therefore, were investigated the morphological characteristics of microglia in these areas of brain. Laser radiation is used to image the dynamic of changing microglia morphological phenotype. An objective of this work is to study the proportion of microglia phenotype and their morphological characteristic after of the middle cerebral artery occlusion (MCAO) and CdEPO treatment, because this is important for assessing the development of ischemic damage. The report presents the results of microglia phenotype distribution in the somatosensory cortex and hippocampus of the mouse brain in the early postischemic period in the MCAO model. For this experiment, we use C57BL/6 mice. Surgery has been made using a binocular microscope and under isoflurane anesthesia. One-hour exposure was proceeded by a 7-0 microfilament from Doccol Corporation. The animal behavior was tested on the first day after the injection. Preservation of basic activity and memory was studied with Open Field LE800S test, Shuttle Box LE916 (PanLab/Harvard Apparatus), "novel object recognition" test. CdЕPO was injected after intravenously in 1, 3, 6, 12, 24, 48 hours after model of transient occlusion of the middle cerebral artery (tMCAO) (one-hour exposure) at 50 mkg/kg dose. Behavior was studied on the 4-th postoperative day. Brain is fixed in 4% paraformaldehyde (PFA). Microglia was marked with primary Iba-1 antibodies (Rabbit polyclonal) and secondary antibody with fluorescent agant AlexaFluor 555. Visualization was carried out by a ZEISS LSM 880 laser scanning confocal fluorescents microscope with an 40x immersion objective and a yellow-green spectrum filter. Paraffin coronal sections of the brain (5 μm thick) were studied. The images were processed using ImageJ and Python 3 software.

Carbamylated darbepoetin is a promising pharmacological agent with universal cytoprotective activity. To assess the acute toxicity, the drug was administered to white laboratory mice and white laboratory rats subcutaneously (from 1000 to 5000 μg / kg) and intravenously (500 to 2500 μg / kg). There was no lethality and no phenotypic changes (including from the red blood sprout) in both species of animals. The study showed that the drug carbamylated darbepoetin (Pharmapark, Russia) belongs to the IV class of low-toxic drugs.

Carbamylated erythropoietin and its analogies have cytoprotecting properties with no erythropoietic activity. Main disadvantage of current technology of carbamylated darbepoetin production is the poor yield. To solve this problem we have tested the influence of such unfolding agents as urea, guanidine chloride, reduced L-glutathione and dimethyl sulfoxide, added at the step of the carbamylation reaction. We have demonstrated the influence of the unfolding agents on both the rate of the reaction and the yield of the whole purification process. The best results were obtained with the addition of 15 % dimethyl sulfoxide - the yield was increased up to 45 %, whereas the influence of the other agents was miserable.

Two licensed vaccines Gardasil and Cervarix, containing main capsid protein L1 virus-like particles antigen, are used for prevention of diseases associated with human papilloma virus. L1 cannot provide protection against all pathogenic strains of HPV, shows low efficacy in middle and old age vaccinated patients, and are not therapeutically effective. The next generation of vaccines that contain minor protein L2 conserved epitopes as well as oncoproteins E6 and E7 promise to provide broad protection against all HPV, and prevent the emergence and spread of skin lesions.

The recombinant E7 oncoprotein gene of type 16 human papilloma virus (conjugated with the heat shock protein 70 (E7(16)-HSP70)) was expressed in Saccharomyces cerevisiae yeast cells. A number of chromatographic isolations were conducted to obtain the target protein and its primary identification and to establish its originality. The use of metal chelate chromatography was based on selective binding of sorbent active groups with a poly-histidine fragment at C-terminus of the target protein polypeptide sequence, while the use of affine chromatography was based on the association with ATP-binding regions of E7 and HSP70. The degree of purity of the target protein after both procedures did not exceed 70%, indicating the presence of many low-molecular admixtures and products of the target molecule oligomerization. The use of chromatographic purification under denaturing conditions using strong reducing agents allowed isolation of the target protein, the monomer content in which exceeded 97%. The target protein originality was established by immunoblotting and electrophoresis; the correspondence of its primary structure to the declared sequence was demonstrated by mass-spectrophotometry.

A nickel-chelate affinity chromatography has been used to isolate fused proteins of Hsp70-E7 from yeast lysate at the initial stage of the work. The low purity of the monomeric Hsp70-E7 in the final fraction was associated with the occurrence of dimeric and tetrameric forms of the target protein and low-molecular impurities. A modification of the purification procces by the addition of reduced L-glutathione to inhibit the formation of unwanted disulfide bonds permitted to increase the purity of the target protein by reducing the content of the oligomerized products. Further changes in the purification protocol, namely the reduction of hydrophobic interactions by the introduction of glycerol, significantly reduced the amount of low-molecular impurities. For a finer purification of E7-HSP70, a two-stage process of anion-exchange chromatography that made it possible to effectively eliminate low-molecular impurities was used. The assessment of the final material by electrophoresis under non-reducing conditions showed that the purity of the monomeric forms of either of the proteins was higher than 95%.

A possibility of biologicals freeze-drying in pre-filled syringes depending on syringe type and various conditions of heat-transfer has been investigated. The effect of preliminary freezing on foaming and lyophilisate quality was studied. An approbation of aluminium blocks destined to the homogenous he- at-transfer within a freeze-dried series was performed. A high- quality product that falls within the existing specifications was oblained.

The approaches to the stabilization of the recombinant fusion oncoproteins E7 16 and 18 conjugated with heat shock protein70 insolutions have been studied. The screening of pharmaceutical active substances was carried out by the accelerated storage test at elevated temperatures. The first stage of the study was aimed at determining of the pH range providing the best stability of the physicochemical quality parameters of the proteins which turned to be 6.8—7.4. The second and third steps were aimed at identification of excipients that make the greatest contribution to the increased storage stability of proteins in solution showed that sucrose and polysorbate 80 constitute the most stable composition. The temperature-20 °Callows to keep the quality parameters on a desired level for at least 12 months.

A gene for a recombinant E7 oncoprotein of human papillomavirus type 16 conjugated with a heat shock protein 70 (E7(16)-HSP70) has been expressed in Saccharomyces cerevisiae. A number of chromatographic separations was performed in order to primarily identify the protein and establish its authenticity. Metal-chelate and affinity chromatography were aimed at selective binding of the active groups of sorbents with polyhistidine fragment at the C-terminus of the polypeptide sequence of the target protein and ATP-binding regions of E7 and HSP70, respectively. The purity of the target protein after two purification steps did not exceed 70% showing the presence of a number of low-molecular impurities and oligomerization products of the target molecule. Chromatographic purification under denaturing conditions using strong reducing agents made it possible to isolate the target protein with the content of the monomer exceeding 97%. Electrophoresis and immunoblotting methods permitted to establish the authenticity of the target protein, and mass spectrometry showed its set primary structure.