ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
License CC-BY.
Biotechnologia Acta Т. 19, No. 3, 2026
P. 32-45 , Bibliography 33 , Engl.
UDC: 615.37:577.27:543.42
doi: https://doi.org/10.15407/biotech19.03.032
EVALUATION OF A TRIS-GLYCINE BUFFER MODIFICATION FOR ANTICOMPLEMENTARY ACTIVITY TESTING OF IMMUNOGLOBULIN G PREPARATIONS
O.V. Yatsenko 1, M.V. Mylenko1, O.M. Dyshliuk1, K. O. Yefymenko1, Y,D. Vinnіchuk2, A,Yu. Labyntsev2,
O,,S. Osypchuk1
1 Biopharma Plasma LLC, Bila Tserkva, Kyiv region, Ukraine
2 Palladin Institute of biochemistry of NAS of Ukraine, Kyiv
Polyvalent human intravenous immunoglobulin (IVIG) is a complex protein preparation obtained from human plasma and requires strict quality control to ensure safety and clinical efficacy. One important quality attribute is anticomplementary activity (ACA), which reflects the potential of immunoglobulin preparations to activate the complement system.
Aim. The study aimed to assess the applicability of the Tris-glycine buffer as an alternative to the pharmacopoeial barbital buffer and to provide practical guidance for laboratories performing ACA testing.
Methods. Тwo approaches for determining ACA were evaluated: the method described in the European Pharmacopeia (Ph. Eur.) and a modified procedure using a Tris-glycine buffer. Тwo approaches for determining ACA were evaluated: the method described in the European Pharmacopeia (Ph. Eur.) and a modified procedure using a Tris-glycine buffer. The tested immunoglobulins were obtained by fractionation of donor blood plasma (de-identified blood samples). Complement activity was evaluated by determining the degree of hemolysis of activated complement. ACA was assayed according to the method described in Ph. Eur. 01/2018:20617 by calculating the ratio of the activity of bound complement in the tested sample (solutions of the preparation and standard samples) to its output activity in the control sample. Data were analyzed using linear regression and a two-tailed Student's t-test (Microsoft Excel).
Results. We tested the Tris-glycine buffer-based modification for the ACA assay. The methodology was effectively reproduced and verified to meet the requirements specified in the Ph. Eur. monograph 01/2018:20617. In addition, the workflow has been optimized, increasing analytical productivity by using deep-well plates instead of individual microtubes, reducing hands-on time, and improving reproducibility by minimizing operator-dependent variability. Ноwever, when barbital buffer is replaced with a Tris-glycine buffer, samples must be adjusted to pH 7.0 before analysis, as the buffering capacity of Tris-glycine is insufficient to maintain a stable pH in the reaction mixture. Under these conditions, all tested samples met the acceptance criteria, with ACA values below 50% (less than 1 CH50/mL per 1 mg of immunoglobulin), indicating minimal complement activation.
Conclusion. The proposed modification represents a practical optimization of the known method, the AСA assay. It facilitates more efficient and reproducible testing in quality control laboratories, while maintaining compliance with established safety criteria for immunoglobulin G preparations.
Keywords: anticomplementary activity, immunoglobulin preparation, quality control.
O. V. YATSENKO https://orcid.org/0009-0009-6060-4870
M. V. MYLENKO https://orcid.org/0000-0002-2662-2137
O. M. DYSHLIUK https://orcid.org/0000-0002-1841-0716
K. O. YEFYMENKO https://orcid.org/0009-0001-2347-5389
Yu. D. VINNІCHUK https://orcid.org/0000-0002-3148-8067
A. Yu. LABYNTSEV https://orcid.org/0000-0002-1793-4630
O. S. OSYPCHUK https://orcid.org/0009-0000-9224-7577
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