INTRODUCTION. A key factor in the creation of biotechnological medicinal products is to establish cell lines for high-yield production of recombinant proteins. The development of selection protocols and highly efficient screening approaches for cell lines producing target proteins is a necessary step in the development of recombinant technology for high-yield target protein production.

AIM. This study aimed to derive producer cell lines from a CHO suspension cell line for high-yield production of the recombinant monoclonal antibody denosumab.

MATERIALS AND METHODS. A CHO-K1 suspension cell line was cultured using serum- and animal component-free media and feeds. The cells were transfected with plasmids containing light and heavy chains of denosumab by electroporation using a MaxCyte STX system. The transfected cells were selected under antibiotic pressure (hygromycin and geneticin). Monoclonal cell lines were obtained using a ClonePix FL system. Leader monoclonal cell lines were identified by determining denosumab concentrations by enzyme-linked immunosorbent assay (ELISA) following fed-batch culture.

RESULTS. The optimum concentrations of antibiotics for the selection of CHO-derived denosumab-producing cell lines were 600 mg/L for hygromycin and 600 mg/L for geneticin. The selection process following transfection was successful in 1041 (about 54%) of 1920 minipools. Denosumab-producing minipools were identified by screening culture fluid samples from 96-, 24-, and 6-well plates using ELISA. Then, 23 leader minipools were chosen and adapted to suspension culture in shaker flasks. The growth and production characteristics of these 23 minipools indicated the leader minipool for cloning (mp-19). This minipool provided a denosumab yield of 1.92 g/L on day 7 of fed-batch culture. Using mp-19, the authors obtained monoclonal cell lines providing up to 6.5 g/L denosumab yields on day 9 of fed-batch culture.

CONCLUSIONS. The authors obtained monoclonal cell lines for high-yield denosumab production. The offered approach to producer cell line development can be applied to the production of various recombinant proteins, including monoclonal antibodies, enzymes, and blood coagulation factors.

INTRODUCTION. The lack of systematic research comparing promoter activity in Saccharomyces cerevisiae limits approaches to improving the efficiency of recombinant human proinsulin biosynthesis. This study compared the activity of constitutive promoters under standardised technological conditions to identify the most productive regulatory elements for application in industrial strains.

AIM. This study aimed to compare the influence of TEF1, ADH2, ALD4, TDH3 (GPD), and TPI1 promoters on recombinant proinsulin expression in Saccharomyces cerevisiae and rank their effectiveness for selecting a highly productive promoter.

METERIALS AND METHODS. S. cerevisiae YS3 diploid strain obtained by haploid cells fusion was used for proinsulin expression. The vectors based on the pRS425 plasmid consisted of synthetic promoters (TPI1, TEF1, ADH2, ALD4, TDH3) and signal sequence. Plasmids were electroporated in the cell. Comparative expression analysis was performed by cultivating S. cerevisiae YS3 in flasks and bioreactors using YPD media. Proinsulin and ethanol concentration was measured using spectrophotometry, HPLC, and gas chromatography. The expression was confirmed using HPLC-MS. Statistical analysis included variance and correlation analysis.

RESULTS. Strains S. cerevisiae YS3/pF1145   (TPI1),   YS3/pF1157   (TEF1),   YS3/pF1199   (ADH2), YS3/pF1200 (ALD4), and YS3/pF1201 (TDH3) were obtained, then expressions of recombinant protein were compared. The strain YS3/pF1201 that had proinsulin expression controlled by the promoter TDH3 showed a maximum productivity of 15.51±0.57 mg/L. When fermented in laboratory bioreactors, the strain YS3/pF1201 achieved a productivity of 139.17 mg/L and a biomass of 154.5 mg/L after 72 hours of cultivation.

CONCLUSIONS. Analysis of experimental data ranked the promoters for proinsulin expression in S. cerevisiae cells by their effectiveness: TDH3≈ALD4>ADH2>TEF1>TPI1. During the fermentation, TDH3 promoter showed significantly higher productivity, which was 6.1 times more than in the classical strain with the TPI1 promoter for proinsulin expression.

INTRODUCTION. Romiplostim treatment of patients with idiopathic thrombocytopenic purpura is associated with formation of anti-drug antibodies (ADA), which often leads to the serious adverse events. A method based on biolayer interferometry is proposed for determination of binding (total) ADA (bADA) to romiplostim in human serum. From a technological point of view, instruments using this principle have some advantages (higher throughput, prolonged service intervals, low level of equipment contamination during analysis of biosamples) over those based on surface plasmon resonance, which are used to monitor bADA levels during clinical trials of the original drug. The method should be validated to ensure reproducibility of measurements and standardise clinical laboratory trials of the immunogenicity of a biosimilar drug.

AIM. To establish the key validation characteristics of a biolayer interferometry-based method for the determination of total anti-drug antibodies to romiplostim in human serum.

MATERIALS AND METHODS. The method is used to detect the specific interaction of binding antibodies with biotinylated romiplostim immobilised on the streptavidin-coated biosensors. The method includes a screening test to determine bADA presence or absence in the samples; a confirmatory test to check the specificity of bADA binding to romiplostim; determination of antibody titer and maximal dilution, that allows to detect bADA in the samples. Positive control samples containing different concentrations of polyclonal rabbit antibodies to romiplostim were used in the work. Detection of protein complex formation was performed in real time mode using an Octet® QKe interferometer.

RESULTS.  The  biological  variability  factor  and  the  limit  of  not  significant  inhibition  were 1.481 and 34.2%, respectively. The precision and specificity of the method were confirmed. The lower limit of bADA detection in the absence of romiplostim was 622 ng/mL. The analytical signal of 8 out of 10 blood serum samples after the addition of bADA increased by more than 1.481 times when assessing the matrix effect in the screening test; the percentage inhibition value exceeded 34.2% when analysing 8 out of 10 samples with the addition of bADA in the confirmatory test. There was no high-dose effect at bADA concentrations from 0.8 to 50 μg/mL. The results of bADA determination in the screening test were robust to the presence of 1 ng/mL romiplostim in the samples. Streptavidin-coated biosensors with immobilised romiplostim maintained stability after 14 days of storage at a temperature of (5±3) °С and at least 20 regeneration cycles.

CONCLUSIONS. Analysis of the validation results obtained confirms the suitability  of  the method for reliable and reproducible determination of binding (total) antibodies to romiplostim in human serum.

INTRODUCTION. Aflibercept is used to treat neovascular age-related macular degeneration, a leading cause of vision loss. Immunogenicity is one of the significant adverse effects that results in the production of anti-drug antibodies (ADAs). The development of a method for detecting ADAs to aflibercept is essential for immunogenicity assessment.

AIM. This study aimed to develop and validate an analytical method for detection and stepwise characterisation of total anti-drug antibodies to aflibercept in human serum utilising the enzyme-linked immunosorbent assay.

MATERIALS AND METHODS. The method was developed using a solid-phase ELISA. Aflibercept (GNR-098, JSC GENERIUM, Russia) and a conjugate of GNR-098 with horseradish peroxidase were employed. Polyclonal antibodies against GNR-098 were obtained by immunising rats, followed by serum collection and antibody purification via affinity chromatography. ADA detection was performed using labelled GNR-098 in a bridging ELISA. Model samples were prepared by spiking pooled sera from healthy donors with rat polyclonal antibodies. Method validation included the following parameters: cut-off points; precision; limit of detection (LOD); drug tolerance; specificity; matrix effect; minimum required dilution; robustness; reagent stability; hook effect.

RESULTS. An analytical method includes a screening assay for the presumptive detection of ADAs; a confirmatory assay to establish ADA specificity and assay for ADA titer determination and IgE isotyping. The cut-off points for the screening assay and IgE-specific assay were 1.3 and 1.2, respectively; for the confirmatory assay, 15.2%. The LOD of the screening assay was 185 ng/mL. The method demonstrated drug tolerance (interference from soluble target, hrVEGF165) at concentrations up to 2,000 pg/mL. No matrix effects were observed in the screening or confirmatory assays across 13 test samples containing ADAs at 280 ng/mL. The absence of a hook effect was confirmed in both assays using samples with ADA concentrations five times higher than the high-positive control.

CONCLUSIONS. An analytical method was developed for the ELISA-based detection and stepwise characterisation of total anti-drug antibodies to aflibercept (GNR-098) in human serum. The method’s suitability was confirmed through validation. This analytical procedure will facilitate the immunogenicity assessment of aflibercept.

INTRODUCTION. Human immunoglobulin preparations (HIP) used in medicine effectively treat autoimmune diseases, inflammatory diseases, and immune deficiencies and prevent diseases of various aetiologies. Harmonising Russian national quality standards (monograph and general pharmacopoeial monographs) for HIP with world quality standards is one of priorities for improving medical supply in Russia. Current quality standards for HIP need to be revised due to significant differences from the leading world pharmacopoeias in what regards quality assessment.

AIM. This study aimed to systematise and analyse national and international compendial quality control requirements for HIP as part of aligning Russian State  Pharmacopoeia  with  the world quality standards, in order to develop HIP monograph drafts.

DISCUSSION. 33 monographs for HIP have been analysed in several pharmacopoeias: 14 monographs in European Pharmacopoeia (Ph. Eur.) and British Pharmacopoeia (BP), 7 — in Indian Pharmacopoeia (IP), and 12 — in Chinese Pharmacopoeia (ChP). The study has shown the number of quality standards for HIP in certain regional and national pharmacopoeias. Ph. Eur., BP, and IP were found to have no general chapters for HIP, while ChP included 2 general chapters. Ph. Eur. and BP showed the highest number of monographs on specific HIP. Currently, United States Pharmacopoeia (USP) shows no monographs on HIP. Considering a recent trend towards harmonisation of national and regional compendial requirements, the authors have analysed HIP quality requirements from Russian State Pharmacopoeia and Ph. Eur., recognised as the basic pharmacopoeia. A comparative analysis of Russian quality standards has shown differences in very important quality parameters, e.g. prekallikrein activator, antibodies to hepatitis B surface antigen, immunoglobulin А (normal HIP for intramuscular and subcutaneous administration), and antibodies to hepatitis А virus (HIP for intramuscular administration). The study also showed the difference in the existing quality  assessment  approach,  and  the  resulting need to align Russian and international quality requirements for HIP, improve quality assurance, and unify control methods. The completed analysis of compendial quality requirements for HIP was used to prepare the drafts of the general chapter monographs on normal HIP as per administration.

CONCLUSIONS. Comparative analysis of compendial requirements shows the need to harmonise Russian quality standards for HIP with the leading world pharmacopoeias, primarily Eur. Pharm. The developed drafts of the monographs have been aligned with their counterparts around the world and include up-to-date methods of quality assessment. Further HIP standardisation requires new compendial references.

INTRODUCTION. When developing and evaluating new biological products (BP), potential adverse action and complicated analytical procedure are a specific issue. Innovative analytical methods and selection of excipients allow for risk minimisation and enhanced quality control of biological products.

AIM. This study aimed to analyse modern quantitative approaches to BP excipients and assess their prospects in improving laboratory expertise during authorisation and quality compliance testing of a biological product that has been launched into the commercial market.

DISCUSSION. The literature search was performed using SciFinder, PubMed, and eLIBRARY.RU databases. The authors showed data on functional classification of the excipients. Potential adverse effects were analysed, including sucrose nephropathy when using intravenous immunoglobulin preparations containing sucrose; hypoglycaemia; changes in amino acid metabolism; decreased DNA and RNA synthesis, and inhibition of platelet function caused by elevated sodium caprylate in albumin preparations. Analysed methods are based on excipient degradation. Possible solutions have been described (reduction or replacement with another excipient, such as sodium caprylate excluded from the BPs of many manufacturers). Innovative search approaches were used for new adjuvants (MF59, АS01, АS03, AS04, and RC-529) combining immune response inducers and delivery systems. The described mathematical model was used to select excipient composition. The authors analysed literature covering analytical methods for excipient quantitation, including liquid and gas-liquid chromatography and spectrophotometric quantitation methods of the most significant excipients in BPs (amino acids, polysorbates, phenol, phenoxyethanol, benzyl alcohol, and sodium caprylate). Prospects of different analytical techniques were considered (size-exclusion HPLC for evaluation of polysorbate 80, HILIC HPLC for selective quantitation of amino acid components, hydrophilic HPLC with refractometric detection and ion-exchange HPLC with amperometric detection for selective quantitation of carbohydrate stabilisers (sorbitol, mannitol, trehalose, glucose, lactose, sucrose, maltose), as well as gas-liquid chromatography for 2-phenoxyethanol and m-cresol.

CONCLUSIONS. Selecting BP excipients is a complex task that requires a control strategy not only for established concentrations, but also for products of possible degradation. Developing unified analytical methods for excipient quantitation is a priority for quality assurance of BPs authorised and launched into the commercial market.

INTRODUCTION. Microbiological quality control of medicinal products relies on the use of reference test strains with stable cultural and morphological properties. Use of lyophilised test strains standardised by number of viable cells reduces the labour intensity of testing compared to the traditional method of preparing microbial suspensions.

AIM. This study aimed to evaluate reproducibility, homogeneity, and stability of results when assessing growth-promoting properties of nutrient media using traditional preparation method of microbial suspensions and the method based on LYOSHTAMM lyophilised test strains standardised by the number of viable cells.

MATERIALS AND METHODS. Two test strains were used: Salmonella enterica subsp. enterica serovar Abony IHE 103/39 and Staphylococcus aureus ATCC 6538. Compared was the method using standardised lyophilised LYOSHTAMM samples (one batch per strain, standardised by the number of viable cells) and the reference method. The latter uses microbial suspensions prepared in compliance with the Russian State Pharmacopoeia (pharmacopoeial monograph “Microbiological purity”). The study was conducted across seven independent laboratories over three days. A total of 378 individual results were obtained for each LYOSHTAMM batch and 126 results — for the reference method.

RESULTS. When evaluating growth-promoting properties of the media, both test strains based on LYOSHTAMM samples met all acceptability criteria: the number of colony-forming units per plate ranged from 10 to 100; typical cultural and morphological properties and growth time was confirmed. Intralaboratory precision (RSD) ranged from 7% to 21% for LYOSHTAMM method and from 2% to 36% for reference method. The interlaboratory precision for LYOSHTAMM samples was 15–17%, while for reference method, it was 38–45%. Time to visible colonies (≈18 hours) and their cultural and morphological properties showed no significant differences between the methods. Analysis of inter-vial homogeneity showed consistent mean values and relative standard deviations at intralaboratory production control (n=5) and  interlaboratory trials (n=63), confirming the stability of LYOSHTAMM batch (relative standard deviation ≤17%).

CONCLUSIONS. The control method for nutrient media using standardised lyophilised LYOSHTAMM test strains yields results comparable to the traditional method in  terms  of growth and morphology. A key advantage of this method is a significantly improved interlaboratory reproducibility, with a more than two-fold reduction in variability. LYOSHTAMM test strains can be recommended as reference standards for routine microbiological testing and analytical validation.

INTRODUCTION. High-risk human papillomavirus (hrHPV) is a proven etiological factor in prevalence of cervical cancer. Screening with HPV genotyping in women and identifying significant regional virus genotypes is vital for implementation of HPV immunisation programmes.

AIM. This study aimed to examine the prevalence, spectrum, and regional characteristics of hrHPV in women from different districts of Gomel region in 2018–2023, prior to the mass vaccination programme in the Republic of Belarus.

MATERIALS AND METHODS. A total of 11,382 women from Gomel region and the city of Gomel aged 18 to 79 years were examined in 2018–2023. Biomaterial samples (endocervical scrapings) were taken from each study participant for subsequent molecular genetic analysis using polymerase chain reaction.

RESULTS. A total of 14 different HPV genotypes have been detected. The incidence of hrHPV was 9% in the overall population and 9.7% in women of reproductive age. High hrHPV incidence was noted in women of early reproductive age: group of 18–24 years — 18.8% (95% CI 16.8–20.9), 25–29 years — 12.1% (95% CI 10.3–14.1), and 30–34 years — 9.2% (95% CI 7.8–10.7). A single HPV genotype was detected most frequently (78.6%); a combination of two HPV genotypes was detected in 86 patients (14.5%); ≥3 genotypes — in 41 patients (6.9%). The most common genotypes were 16 (52.2%), 18 (15.1%), 51 (18.9%), 56 (9.8%), and 31 (9.7%) HPV. Groups α9 (16 and 31 genotypes), α7 (18 genotype), α5 (51 genotype), and α6 (56 genotype) most frequently caused hrHPV infection in women. A change has been found in the structure of dominant hrHPV genotypes in Gomel region over the past 10 years.

CONCLUSION. A high hrHPV prevalence has been established among women of reproductive age in Gomel region. Data on HPV genomic diversity in the region are essential for molecular epidemiological surveillance and will help assess the effectiveness of the Republican HPV immunisation programme launched in 2025.

INTRODUCTION. During the COVID-19 pandemic, acute respiratory distress syndrome (ARDS) was diagnosed in 15–33% of patients hospitalised for pulmonary diseases. Hospital mortality rates increased. The existing medicinal products lacked effectiveness. Thus unconventional treatment methods were needed, such as mesenchymal stromal cell (MSC) therapy. The risk of blood clotting in the lung vessels after MSC injection made exosomes from MSC secretome a therapy of choice. Exosomes cross the blood-brain barrier and have regenerative effect similar to that of MSC. The promising results of preclinical trials for exosome-based drugs have stimulated their clinical use. Analysing their safety and effectiveness will allow us to develop protocols for their production, storage, and transportation, as well as optimal dose regimens for cell-free therapy of ARDS and other pulmonary diseases.

AIM. This study aimed to analyse performed preclinical and clinical studies on safety  and efficacy of MSC-derived exosome drugs intended for cell-free ARDS therapy and other pulmonary diseases as an alternative to drug therapy.

DISCUSSION. Exosomes, the most important secretome element in various cells, carry out horizontal transfer of genetic information and bioactive molecules. Animal models show that exosomes obtained from MSC secretome have regenerative abilities similar to MSC and offer various advantages: small size excluding blood clotting in the pulmonary capillaries; ability to penetrate blood-brain barrier, non-teratogenicity, and exchange of epigenomic information in cell-cell interactions. Preclinical in vivo studies have shown that exosomes affect regeneration of damaged lung tissue in ARDS and other lung diseases. Clinical trials have confirmed safety and effectiveness of inhalation, intravenous or combined administration. Drug effectiveness can be increased by combining exosomes with MSC or enriching them with CD24 (key molecule of innate immunity). Due to regenerative, immunomodulatory properties of exosomes, their ability to reduce the level of cytokine storm and apoptosis, they are used to treat ARDS and other lung diseases. Exosome preparations reverse ARDS and other diseases due to their regenerative and immunomodulatory effect, and ability to reduce cytokine storm and apoptosis. Thus exosomes are recognised as a new effective cell-free therapy.

CONCLUSIONS. Therapeutic effect of exosome-based preparations was analysed in experimental, preclinical, and clinical trials; however, further trials are required to determine ARDS safety and optimal treatment regimens.