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. 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 stan dardise 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.