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.

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

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.