Upon the development of biosimilar drugs, the structural, physical-chemical, and functional similarity to the original drug should be ensured. We have proposed a simple and rapid HPLC-UV method for assessing the content of C-terminal Arg166 in darbepoetin alfa samples, in particular after their isolation from the culture fluid. All the stages of the described method (from the sample extraction to obtaining a chromatographic profile) take about 5 h. The proposed approach will be useful for laboratories developing a technology for producing recombinant darbepoetin alfa, to assess the quality of the product, and for subsequent optimization of the production process.

A number of drugs based on recombinant erythropoietin contain human serum albumin as an auxiliary component. The presence of this protein hinders the proper control of the drug quality in accordance with the requirements of regulating agencies. We propose the novel method for separation of recombinant erythropoietin (epoetin beta) and human serum albumin. It is based on the subsequent use of hydrophobic sorbent and anion exchange resin placed in gravity flow columns (without the use of spin-columns). The proposed approach makes it possible to concentrate and purify the preparations containing the epoetin beta both at high and at minimal concentrations (the ratio of the amount of albumin and erythropoietin in the used preparations can reach 125:1). The average yield of epoetin beta after the use of hydrophobic sorbent and anion exchange resin was 75% and 97%, respectively. It was shown that the determined conditions of sample preparation had no affect on the content of the epoetin beta in the product.

BACKGROUND:Owing to improvement of the molecular diagnostic methods using purified preparations of nucleic acids (NAs), the development of effective methods providing the isolation of DNA is still relevant. The sorption properties of magnetic multi-walled carbon nanotubes (MWCNTs), oxidized MWCNTs and MWCNTs (pristine and oxidized) modified with polydiallyldimethylammonium chloride (pDADMAC) with respect to double strained DNA have been studied.RESULTS:It was shown that in the presence of MWCNTs/pDADMAC particles the DNA molecules were reversibly retained by the particle's surface. The optimal conditions for each step of DNA extraction from model solutions using the obtained material were selected. A comparative evaluation of the effectiveness of the proposed method for DNA isolation based on the results of spectrophotometry and real-time PCR was carried out. It was shown that the desorbed DNA was efficiently amplified in PCR, inhibition of polymerase did not occurred. Probable mechanisms of DNA retention due to the influence of residual impurities of catalysts in the MWCNT composition, as well as the surface charge of nanotubes are proposed.CONCLUSION:Sequentially oxidized and coated with pDADMAC magnetically susceptible CNTs are seemed to be a promising material for development of low-cost systems proving an easy isolation, storage, and subsequent use of dsDNA in molecular diagnostics. The sorption properties of such systems are determined with highly developed specific surface area and their chemical composition.

Objectives. Polysorbate 80 (PS80) quantification in biopharmaceutical products has always been challenging owing to its minute content, absorption to the protein backbone, lack of specific chromophoric PS80 groups, and heterogenic nature. This work is aimed at developing an express method for PS80 analysis in biopharmaceutical products using hydrolysis and subsequent highperformance liquid chromatography analysis with ultraviolet detection that does not consume substantial amounts of sample (≥35 μL).Methods. Five therapeutic protein formulations were chosen as model proteins. Alkaline hydrolysis formulation was applied, without protein precipitation and with a range of precipitation techniques to remove protein from the test solution and hydrolyze PS80, to free fatty acids. The obtained hydrolysate was analyzed using reverse-phase high-performance liquid chromatography.Results. As a result of the high protein content of monoclonal antibody formulations, preliminary protein removal was required, which was achieved by precipitation with organic solvents. A specific precipitant ethanol–isopropanol mixture (1:1 volumetric ratio) was developed to efficiently remove antibodies while keeping PS80 in the solution. The PS80 quantification method was developed for monoclonal antibody drugs. For three monoclonal antibody drug products (adalimumab, infliximab, and eculizumab), method validation was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use, the United States Pharmacopeia, and the State Pharmacopeia of the Russian Federation guidelines.Conclusions. The optimal assay conditions for each group of recombinant monoclonal antibody substances were chosen. Protein precipitation with ethanol or ethanol–isopropanol mixtures before hydrolysis was introduced, allowing for a substantial reduction of sample to 35 μL or even less if PS80 content is higher than 0.05 mg/mL. Accelerated hydrolysis (90 min) is preferable to slow hydrolysis (4–18 h). Method validation for protein products such as adalimumab, infliximab, and eculizumab was demonstrated for the first time. Both methods were validated for each drug product. The coefficients of variation for method specificity and high precision were ≤6.0% for 3 analyses. The accuracy of the methods ranged from 96% to 109% for all of the tested drug products.

The method providing one-step isolation of DNA from soil extracts was developed. Isolation of nucleic acids from the biological objects presenting in a soil usually is carried out by in situ lysis of the sample followed by DNA purification. The purification step is complicated by the fact that about 60% of all soil organic substances are humic substances (HSs), which are powerful inhibitors of the polymerase. The currently known methods of DNA isolation from soil samples are usually laborious and multistage. In our opinion, a promising approach to solve this problem could be based on the use of a two-component bioseparating element. The first component of such element is a special sorbent demonstrating the effect of "negative selection" in relation to DNA (when DNA is not retained by the sorbent while the admixtures are effectively held), and the second polymeric component exhibits high selectivity in binding of HSs. The effectiveness of the use of such two-component system for one-step isolation of DNA from soil extracts with simultaneous purification of DNA from HSs was confirmed by spectroscopic, electrophoretic, and chromatographic methods. The developed element was represented as a compact spin-cartridge containing two layers of different sorbents. In particular, polyaniline-modified silica effectively retained proteins, and polymer alginate particles provided the retention of hydrophobic (similar to 98%) and hydrophilic (>75%) fractions of HSs. The both used components did not retain DNA. Achieved purity of the isolated DNA provided the possibility of its direct use in PCR analysis. The yield of purified DNA estimated by the model experiments was more than 90% .

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.

The review considers the role that nanotechnologies play in the development of sample preparation methods for molecular diagnostic, focusing on the methods to isolate nucleic acids (NAs) from biological samples and the underlying physicochemical processes. Methods based on reversible adsorption (that is, solid-phase selective extraction) are the most efficient and allow miniaturization and automatization of the related processes. In the approach most commonly used until recently, NAs from biological samples are bound with a sorbent and then eluted (positive selection). The review analyzes the potential and advantages of an alternative one-step NA isolation method. Sorbents utilized in the method bind proteins and other components of biological samples, but are inert towards NAs in terms of absorption (negative selection). Consideration is given to the methods used to produce nanostructured composite sorbents on the basis of solid matrices (porous silica, glass multicapillaries, and synthetic membranes) via modification with nano-thick polymer layers to achieve negative selection toward NAs. Primary attention is payed to fluoropolymers and polyanilines, their applications, and fields of their alternative use.

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

An HPLC procedure was developed to determine twenty proteinogenic amino acids, ammonium, cystine, and alanyl-glutamine dipeptide in liquid culture media. 6-Aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC) was used as a fluorescent label for the derivatization of amino acids with the subsequent HPLC separation on a reversed-phase column. Specificity, linearity, correctness, precision, analytical range, and limit of quantification were evaluated for each particular component in the procedure validation tests. The analytical range for amino acids was 10–800 μM (20–800 μM for tryptophan and cystine), and the limit of quantification was no higher than 5 μM (15 μM for tryptophan and 8 μM for cystine).

Objectives . We hereby describe an improvement of a previously developed quantification technique for polysorbate 80 in biopharmaceutical formulations (darbepoetin alfa and eculizumab) and report the validation of the new approach. Methods . Polysorbate was isolated from analyte samples by protein precipitation using an organic solvent, followed by supernatant evaporation in vacuum. Polysorbate was derivatized using a ferric thiocyanate reagent and extracted into an organic phase; the relevant optical density measurements were performed. Results . We established the optimal conditions for each step of the analysis procedure. The accuracy was 97–102% in the tested analytical range, the relative standard deviation did not exceed 5%, and the limit of quantification was 0.01 mg/mL. Conclusions. The reported approach is highly sensitive; polysorbate isolation and quantification do not depend on the matrix or, most importantly, the protein.

Objectives. We hereby describe an improvement of a previously developed quantification technique for polysorbate 80 in biopharmaceutical formulations (darbepoetin alfa and eculizumab) and report the validation of the new approach.Methods. Polysorbate was isolated from analyte samples by protein precipitation using an organic solvent, followed by supernatant evaporation in vacuum. Polysorbate was derivatized using a ferric thiocyanate reagent and extracted into an organic phase; the relevant optical density measurements were performed.Results. We established the optimal conditions for each step of the analysis procedure. The accuracy was 97–102% in the tested analytical range, the relative standard deviation did not exceed 5%, and the limit of quantification was 0.01 mg/mL.Conclusions. The reported approach is highly sensitive; polysorbate isolation and quantification do not depend on the matrix or, most importantly, the protein.

Objectives. Developing reliable and accurate analytical methods is necessary for comparative pharmaceutical analysis using physicochemical, biological (in vitro), preclinical, and clinical trials. The main objective of this study was to develop and validate an in vitro method for determining the specific activity of the recombinant monoclonal antibody eculizumab.Methods. The method of indirect enzyme immunoassay was used in the study.Results. A method for determining the specific activity of the humanized recombinant monoclonal antibody eculizumab was described and validated for the first time. A comparative evaluation of the specific activity of Soliris® (Alexion Pharmaceuticals Inc., USA), and its biosimilar PRK-001 (Pharmapark, Russia) was performed using the developed method.Conclusions. The similarity of PRK-001 and the original Soliris® in relation to their specific activity, that is, binding to the human complement system C5 protein, was proved.

Drug elution profiles must be studied in vitro to optimize a polymer-drug formulation during development of drug-eluting stents (DESs). Results from HPLC assays of drug contents and elution kinetics from a biodegradable sirolimus coating and a stable zotarolimus coating on coronary DESs are presented. Drug contents were assessed for crimped stents on the delivery system and expanded stents. The drug coating morphology and elution kinetics were demonstrated to be associated. Significant coating morphological defects were shown to cause deviations in the drug elution profile.

Polyaniline (PANI) and polyaramides deposited on the surfaces of glass slides and particulate silica were studied as adsorbents of nucleic acids and proteins by flow-through spectral correlation interferometry and solid-state extraction using spin-cartridges. Double stranded DNA from E. coli as well as pepsin, bovine serum albumin and lysozyme were the analytes studied in contact with the polymer nanolayers in phosphate buffer solution, pH 7.2. None of the coated glass slides could bind the DNA, which passed them practically without adsorption. In contact with polyaramides, the proteins of pI > 4 reversibly formed the 0.2-2.5 nm-thick adsorption layers decomposing on further rinsing with the protein-free eluent. In contact with PANI, the proteins formed stable adsorption layers at pH 7.2, which needed the pH 3.0 to be eluted. Thus, in a neutral aqueous medium optimal for separation of biopolymers, polyaramides, although did not retain DNA, had a weaker affinity to proteins as compared to PANI. Since the recovery of DNA passed through the PANI-coated silica was the maximal among the particulate adsorbents, the PANI-modified composites were preferred as the carriers for the single-step isolation of nucleic acids from complex biological mixtures. (C) 2017 Elsevier B.V. All rights reserved.

Sorption properties of composite silica sorbents modified with nanolayers of fluoropolymers; polyanilines and polyaramides containing aromatic nitrogen; fluorine; as well as donor and acceptor fragments; with respect to nucleic acids (DNA and RNA) and proteins differing in molecular weight and pI are considered. The use of the investigated sorbents in the sample preparation for molecular diagnostics (in particular; in the PCR analysis) not only provides a one-step isolation of nucleic acids; but also allows the isolation and simultaneous purification of protein compounds from the impurities presented in the initial mixture. For the first time; the properties of these materials are compared in the static sorption regime using the compact spin-columns and in the regime of dynamic sorption by the method of spectral-correlation interferometry. The effect of the chemical composition; morphology; and surface charge of these polymer coatings on their sorption properties was studied. Possible mechanisms of sorption of biopolymers on the investigated sorbents are discussed. The use of the developed approaches to the analysis of properties of the sorbents as well as the obtained data open new possibilities for the synthesis of composite sorbents with specific properties. PANI and polyaramides were shown to demonstrate the similar sorption properties when interacting with nucleic acids; but they differ in a various extent in the retention of various proteins. In a neutral aqueous medium (optimal for separation of biopolymers) polyaramides; although did not retain DNA; had a weaker affinity to proteins as compared to PANI. Since the recovery of DNA passed through the PANI-coated silica was the maximal among the particulate adsorbents; the PANI-modified composites were preferred as the carriers for the single-step isolation of nucleic acids from complex biological mixtures. The article cites the results of systematic studies of the authors in the development of sorbents for one-step separation and isolation of biopolymers from complex biological mixtures.Forcitation:Liaw D.-J.; Zybin D.I.; Prostyakova A.I.; Yagudaeva E.Yu.; Vikhrov A.A.; Ishchenko A.A.; Zubov V.P.; Kapustin D.V. Static and dynamic sorption of nucleic acids and proteins on surface of sorbents modified with nanolayers of polymers. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. 2018. V. 61. N 1. P. 4-22

The effect of chemical composition and morphology of the surface layers of new polyaramide-containing sorbents on the mechanism of selective sorption of nucleic acids and proteins was investigated as compared to the previously studied sorbents modified with fluoropolymers and polyaniline (high-throughput materials providing one-step isolation of DNA from biological mixtures). A series of silica-based sorbents modified with polyaramides having consistently varying structure and containing the set of "key" structural elements (aromatic units and nitrogen atoms in the backbone, fluorinated groups), and various donor and acceptor moieties was prepared. The chemical composition of the polymer coatings was evaluated by X-ray photoelectron spectroscopy. The surface morphology was studied by scanning probe microscopy. The sorption properties were investigated by passing the mixtures containing DNA, RNA and proteins of different nature through the cartridges containing the obtained sorbents. All the investigated materials weakly retain double-stranded DNA but effectively retain RNA and proteins. The sorption capacity of the sorbents depends on the protein nature. The observed sorption behavior was shown to be determined by the chemical structure and not by the morphology of the polymer coating. It was proposed that similarity of the sorption properties of the series of chemically different polymers could be determined by similar total input of different sorption mechanisms.