Acta Chimica Slovaca (ACS) publishes papers on fundamental and applied aspects of chemistry, biochemistry, chemical technology, chemical engineering and process control, biotechnology and food technology. Welcome are also topics which include chemical aspects of materials, physical chemistry and chemical physics, analytical chemistry, macromolecular chemistry and biomedical engineering.

A fluorescent dye assay for antibody aggregates and its application in aggregate stability evaluation

Tomáš Molnár, Veronika Rupčíková, Simona Kotuličová, Milan Polakovič *

Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia

E-mail: * milan.polakovic@stuba.sk

Abstract: Aggregation of proteins naturally occurs during different stages of cultivation and downstream processes and is often associated with quality reduction in the final product. Therefore, determining the presence of aggregates is important. In this study, a fluorescence-based analytical method using the dye Nile red was developed for antibody aggregates quantification. The fluorescence assay demonstrated selectivity and sensitivity towards hydrophobic surfaces of aggregates within reliable quantification range of 0.0165—1.65 g/L. Validation confirmed high reproducibility, with intra-assay and inter-assay coefficients of variation within acceptable limits. The dye was stable in commonly used buffer systems and in the presence of salts. Comparative analysis of SEC and fluorescence methods indicated that fluorescence detection remains sensitive to aggregation beyond SEC’s detection threshold. Thermal stability studies revealed that polyclonal antibodies have higher resistance to thermal stress than monoclonal antibodies, which suggests that structural variability plays an important role in thermal stability. Additionally, antibody aggregation was shown to be pH-dependent, with increased aggregation at both low and high pH levels. Polyclonal antibodies were stable at pH 5—6. In more acidic conditions, soluble aggregates were formed, while at neutral and basic pH, aggregation was accompanied by the formation of insoluble particles. The effect of salts on aggregation was variable, with NaCl promoting aggregation at high concentrations, NaSCN facilitating dissociation of aggregates, and Na2SO4 inducing precipitation. Guanidine HCl exhibited a stabilizing effect, preventing aggregation over time.

Keywords: monoclonal antibody, polyclonal antibody, fluorescence assay, aggregation, kinetics

Acta Chimica Slovaca, Vol. 18, No. 1, 2025, pp. 41—47, DOI: 10.2478/acs-2025-0005