INTRODUCTION. In the absence of an international reference standard for the antigen and the corresponding antiserum for quantifying influenza virus hemagglutinin (HA) content by single radial immunodiffusion (SRID), as well as during pandemics or other emergencies, validated alternative methods for HA quantification are required.

AIM. Optimization of the method for certifying a primary reference standard for quantification of influenza virus hemagglutinin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by densitometric analysis of the gels.

MATERIALS AND METHODS. The HA content (%) in the reference standard was determined by SDS-PAGE under reproducibility conditions (two laboratories). HA concentration (µg/mL) was calculated from the total protein content determined by the Lowry method. The SRID method was used to evaluate the accuracy of HA quantification. The CombiStats software was used for plate image processing, calculation of the squared diameters of precipitation rings, and determination of HA content in the samples. Sucrose and glycerol in the virus concentrate and the diluted virus concentrate (DVC) were quantified by high-performance liquid chromatography.

RESULTS. When performing SDS-PAGE with deglycosylation, the addition of 0.072 mM 2-mercaptoethanol for sample processing eliminates the need for urea. The HA concentrations in DVC determined by SDS-PAGE using 4–12% Bis-Tris gels in Laboratory 1 (135.8 µg/mL) and 4–20% Tris-glycine gels in Laboratory 2 (135.5 µg/mL) were virtually identical; the coefficient of variation did not exceed 4% in either laboratory. The HA concentration determined by SDS-PAGE in Laboratory 1 was comparable to the SRID data (123.6 µg/mL). The content of excipients (glycerol and sucrose) in DVC did not exceed 55 µg/mL and did not affect the results of protein determination.

CONCLUSIONS. Optimal SDS-PAGE conditions were established for certifying a primary liquid reference standard for quantification of influenza virus HA, yielding results comparable to those of the SRID method. The next phase of the study will involve testing the sample preparation conditions and quantification methods for all HA types (H1N1, H3N2, and B) relevant to the 2025–2026 season.

INTRODUCTION. Glycan profile is a critical quality parameter for therapeutic monoclonal antibodies (mAbs) that is consistently estimated during development and release of each drug batch. The need to develop a reproducible sample preparation protocol for glycan profiling brings about the relevance of the study. The profile should be adapted to the conditions of a standard physicochemical laboratory, so that it avoids using commercial preparation kits that are currently in short supply.

AIM. This study aimed to develop an alternative sample preparation procedure for quantitation of glycans in therapeutic monoclonal antibodies without using commercial kits.

MATERIALS AND METHODS. Omalizumab, ustekinumab, canakinumab, tocilizumab, natalizumab, and human anti-PD IgG2 were used as the study objects. N-glycans were released from mAbs by peptide-N-glycosidase (PNGase F) and labeled with fluorescent tags 2-aminobenzamide (2-АВ) or 2-aminobenzoic acid (2-АА) or InstantAB. Subsequently, glycan samples were cleaned from impurities. Glycan compounds were analyzed using hydrophilic interaction liquid chromatography (HILIC-FLD) on Alliance e2695 and Acquity Arc Bio chromatographic systems equipped with FLR 2475 fluorescence detector. The glycan profile was assessed by the content of functional glycan groups.

RESULTS. Conditions for preparing therapeutic mAbs N-glycans were chosen as follows: incubation with 2 mEU PNGase F per 100 µg protein in 10 mM Tris-HCl (pH 8.0) at 37 °C for 3 h without protein denaturation; derivatization of glycans with 2-AA at 65 °C for 1.5 h; extraction of 2-AA excess with acetonitrile. Centrifugation of labeled glycans with acetonitrile is suitable for purification and concentration of samples. Minor glycans with a content not more than 0.1–0.2% were determined using HILIC-FLD. Comparison with data obtained using a commercial kit for sample preparation indicated acceptable comparability of the results. When preparing the samples, we took into account structural features of specific mAbs, for instance, desialylation control during the staining was found necessary for high-sialylated mAbs.

CONCLUSIONS. The developed sample preparation procedure is suitable for the analysis of N-glycans of omalizumab, canakinumab and natalizumab and may be used to develop the analysis methods of the glycan profile of other glycoproteins using HILIC-FLD mode.