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.

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).

A strategy for optimization of the glycosylation profile of Adalimumab, a monoclonal antibody to TNF6 produced by CH O culture has been described. Adalimumab is an analog of a monoclonal antibody that serves as a substance of the Humira preparation, a product of Abbot GmbH & Co. KG (USA). Culturing in a closed volume with periodical feed addition (fed-batch regime) was the basis of the developed technology. A great number of media and feeds from various manufacturers were tested. All of them contained no components of animal origin and supported the CHO cell line suspension cultivation. The analysis of separated from the antibody oligosaccharides and their comparison with the original preparation were performed by chromatomass-spectrometry. Three lots of the obtained preparation were analyzed, and it was shown that they all contained the typical glycans for antibodies produced by the CHO cells: the complex fucosylated G0F/G1F/G2F and nonfucosylated G0/G1 forms, and also glycans with 5 mannose residues (Man5). The ratio of glycans was also typical for antibodies obtained by CHO cells culturing: the predominating forms contained fucose rather than galactose. Based on the analysis of the glycosylation profile and data on antibody productivity, a combination of Dynamis (Gibco, USA) medium and ActiCHOA/B (GE Healthcare, Austria) feed that provided the antibody productivity of 1.58 g/L during fed-batch culturing was selected for the further investigation. It was established that the galactose (19.6% in the obtained versus 23.5% in the original preparation) and fucose (85.7% versus 88.7%) content were similar in both preparations, i.e. the amounts of main glycans and monosacharides as their components are close to the desired status. The significant difference was only observed in the content of a minor glycan, Man5 (1.7% versus 6.3%). The importance of those differences can be assessed in two ways: on one hand, they are beyond the permissible limits of variability of the original preparation; on the other, they were observed at an early stage of the technology, during a small-volu-me culturing.

A combination of cultural medium and nourishment providing a high yield of target glycated forms of recombinant darbepoetin alfa produced by CHO cell culture was selected. The influence of different substances-precursors of glycans in glycosylation reactions-on the yield of highly sialylated forms of recombinant darbepoetin alfa was researched. It was shown that the use of 20 mM N-acetyl-D-mannosamine is the most effective for an increase in the concentration of highly sialylated forms of the protein in cultural fluid.

The selection of medium—feed combination for the improvement of the target glycoforms yield of recombinant darbepoetin alpha produced by CHO cell culture has been performed. The impact of various glycan precursors in the glycosylation process on the yield of highly sialylated darbepoein alpha forms was investigated. The supplementation of the cell culture with20 mM N-acetyl-D-mannosamine was shown to be most effective for the increase in highly sialylated darbepoein alpha forms content.

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 chemically defined medium Hybris-1-CHO designed for suspension culturing and recombinant protein production by CHO cell lines has been characterized. The two-stage character of cell growth and basic biochemical parameters was demonstrated in both batch and fed-batch experiments with the CHO-S model cell line. The data on the culture growth rate, its morphology, concentrations of key metabolites, and also culturing characteristics of the medium and darbepoetin productivity of the cultures on Hybris-1 -CHO in comparison with a few commercial media permit to regard the designed medium as applicable to maintaining of protein biotechnological production.

A strategy for optimization of the glycosylation profile of Adalimumab, a monoclonal antibody to TNF6 produced by CH O culture has been described. Adalimumab is an analog of a monoclonal antibody that serves as a substance of the Humira preparation, a product of Abbot GmbH & Co. KG (USA). Culturing in a closed volume with periodical feed addition (fed-batch regime) was the basis of the developed technology. A great number of media and feeds from various manufacturers were tested. All of them contained no components of animal origin and supported the CHO cell line suspension cultivation. The analysis of separated from the antibody oligosaccharides and their comparison with the original preparation were performed by chromatomass-spectrometry. Three lots of the obtained preparation were analyzed, and it was shown that they all contained the typical glycans for antibodies produced by the CHO cells: the complex fucosylated G0F/G1F/G2F and nonfucosylated G0/G1 forms, and also glycans with 5 mannose residues (Man5). The ratio of glycans was also typical for antibodies obtained by CHO cells culturing: the predominating forms contained fucose rather than galactose. Based on the analysis of the glycosylation profile and data on antibody productivity, a combination of Dynamis (Gibco, USA) medium and ActiCHOA/B (GE Healthcare, Austria) feed that provided the antibody productivity of 1.58 g/L during fed-batch culturing was selected for the further investigation. It was established that the galactose (19.6% in the obtained versus 23.5% in the original preparation) and fucose (85.7% versus 88.7%) content were similar in both preparations, i.e. the amounts of main glycans and monosacharides as their components are close to the desired status. The significant difference was only observed in the content of a minor glycan, Man5 (1.7% versus 6.3%). The importance of those differences can be assessed in two ways: on one hand, they are beyond the permissible limits of variability of the original preparation; on the other, they were observed at an early stage of the technology, during a small-volu-me culturing.