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SCIENTIFIC RELEVANCE. An important quality-control issue for therapeutic monoclonal antibodies (mAbs) is the determination of residual protein A leaching from the carrier during the purification of mAbs by affinity chromatography. However, unrelated compounds (immunoglobulins) present in the sample can complicate the immunoenzymatic detection of protein A (matrix effect), potentially leading to false-negative test results. To increase the efficiency of enzyme-linked immunosorbent assay (ELISA), it is necessary to develop a sample preparation step that can irreversibly break the bond in the protein A–mAb complex.AIM. This study aimed to develop and validate an analytical procedure for the determination of residual protein A in active substances of therapeutic mAbs by ELISA with a test kit comprising in-house reagents.MATERIALS AND METHODS. Recombinant protein A was used as an antigen. Polyclonal antibodies to protein A were produced by immunising chickens, selecting immunised eggs, and isolating antibodies from these eggs. Protein A-specific antibodies were purified by affinity chromatography. Residual protein A was determined using sandwich ELISA with preliminary sample preparation. The analytical procedure was validated in accordance with the requirements of the State Pharmacopoeia of the Russian Federation (Validation of Analytical Procedures, OFS.1.1.0012).RESULTS. The authors obtained, isolated, and purified chicken IgY antibodies to recombinant protein A. The authors selected sample preparation conditions for the determination of residual protein A by ELISA and optimum compositions of buffer solutions, including the composition of a denaturing buffer to disrupt the protein A–mAb complex. The developed analytical procedure was validated. According to the measurements of protein A, the accuracy of the analytical procedure ranged within 83–108% of the nominal value, the interlaboratory precision ranged within 96–116%, and the repeatability was up to 13%. The lower limit of quantitation was 10 ng/mL with a minimum required dilution of 1:10. The analytical range extended from 10 to 40 ng/mL. The analytical procedure showed results comparable to those obtained with a similar test kit from an international manufacturer.CONCLUSIONS. The developed analytical procedure for the determination of residual protein A by ELISA with a test kit comprising in-house reagents can minimise the matrix effect in therapeutic mAbs. This analytical procedure will alleviate import substitution and reduce quality control costs for Russian immunobiologicals.

Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer. Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients. The development of monoclonal antibodies (mAbs) specific for PD-L1 or PD-1 is a promising area of immunotherapy of malignant tumours. However, before a therapeutic antibody-based product enters the market, it is necessary to ensure its safety and efficacy, i.e. perform a full scope of preclinical and clinical studies. The aim of the study was to develop and validate a bioanalytical method that does not require additional labeling and that could be used for determination of mAbs specific for human PD-L1 in the blood serum of a biological test system during preclinical studies. Materials and methods: an antigen in the form of a dimer of PD-1 extra-cellular domain covalently bonded to the Fc-fragment of human IgG (R&D Systems, USA) was used in the study. The antigen was immobilised on Dip and Read™ Protein A biosensors (Fortebio, USA). The therapeutic anti-PD-1 antibody GNR-051 was developed and produced by IBC “Generium” (Russia). The healthy cynomolgus monkey serum samples used as matrix were obtained from the Research Institute of Medical Primatology (Sochi, Russia). The assessment of binding was performed using Octet® QKe interferometer (Fortebio, USA) by real-time analysis of the dose-dependent rate of the antigen-antibody complex formation. Results: the paper presents experimental data on the development and validation of the test method for determination of the therapeutic PD-1-binding mAb concentration in cynomolgus monkey serum in the antibody concentration range from 2 to 2500 µg/mL. The authors assessed the calibration curve reliability, between-run and within-run precision and accuracy, dilution linearity, specificity and selectivity of the test method. Conclusions: the authors developed and validated the biolayer interferometry-based method for determination of therapeutic mAbs concentration. The method was shown to comply with the Eurasian Economic Union’s regulatory requirements in terms of the main validation parameters: analytical range, accuracy, precision, and selectivity.

Enzyme replacement therapy (ERT) is one of the most efficient treatments for lysosomal storage diseases. Type 1 Gaucher disease is caused by β-glucocerebrosidase enzyme deficiency, which may be compensated for by intravenous infusions of imiglucerase—a recombinant enzyme. Imiglucerase targets macrophages and enters these cells via interaction with mannose receptors on the cell membrane. Characterisation of internalization of enzymes by target cells is important in the context of the development of new medicines and production of existing ERT medicines. The peritoneal and alveolar macrophages, as well as macrophages of the spleen of small laboratory animals (rats and mice) are widely used in such studies. However, isolation of cells from animal sources raises ethical issues, and therefore continuous mammalian cell lines may offer an attractive alternative. The aim of the study: to conduct comparative studies on the internalization of recombinant imiglucerase into mouse peritoneal macrophages and L929 mouse fibroblasts. Materials and methods: CerezymeR batches 7HV0913, C6214H05, 7HV0888 (Genzyme Ltd., UK); Glurazim batches 020416, 011117, 021117 (LLC “IBC “Generium”, Russia). We used peritoneal macrophages obtained from BALB/c mice and L929 mouse fibroblasts. The cells were cultured in DMEM/F12 complete growth medium with 10% fetal bovine serum. The activity of imiglucerase internalized into the cells was evaluated spectrophotometrically by hydrolysis of the artificial substrate—4-methylumbelliferyl-β-Dglucopyranoside. Results: the study compared internalization of recombinant imiglucerase (the active ingredient of CerezymeR and Glurazim) by mouse peritoneal macrophages and L929 mouse fibroblasts. It was demonstrated that the medicines activity in the lysates of peritoneal macrophages is comparable with that in the lysates of L929 mouse fibroblasts. Regardless of the model system, the activity of Glurazim stayed within the acceptable range (80–125%) established for biosimilar products. Conclusions: the experiments proved that L929 mouse fibroblasts could be recommended for assessment of internalization of recombinant imiglucerase.

Using the example of Herceptin (Roche) a method of assessing the antiproliferative activity of monoclonal antibodies using tetrazolium salt was developed. The method is based on spectrometric measurement of the level of formazan transformed by living cells from a water soluble salt XTT. The level of formazan is proportional to the number of viable cells. The technique was in terms of specificity, calibration curve, accuracy and precision.

Bioanalytical methods are applied at the development and test of drugs as well as at the step of pharmaceutic products issue. Drugs and excipients quality estimation is made by means of precise and reproducible methods. Accuracy and reproducibility of a method is established during validation which is obligatory for medicine registration. The principal task of method validation is experimental evidence of its suitability for the objectives to be achieved. Validation of bioanalytical methods is one of the elements of the whole medicine production validation [1, 2].The aim of research: to validate a method for determination of specific anti-hemolytic activity of eculizumab developed in LCC “IBC Generium”.Materials and methods: eculizumab, antibody-sensitized chicken erythrocytes, complement-containing human serum.Results. We demonstrated the specificity of the method and its correspondence to criteria of accuracy (103.0±1.4)%, robustness (CV – 11.5%), repeatability (CV – (4.9±0.9)%), reproducibility (CV – (3.5±0.4)%), and linearity (k -1.0275; R2 – 0.9975) during validation. The system validity (equipment, materials, analytical operations and analyzed samples) was confirmed for true results obtaining during validation.Results discussion. Experimental evidence of suitability of the method for eculizumab specific activity assessment was obtained in course of validation. The simplicity of the method allows obtaining accurate results in other laboratories. The developed method can be used not only for specific activity of Soliris determination but also for other pharmaceutical substances and drugs based on antibodies specific to human complement C5.