Many biopharmaceuticals, and especially monoclonal antibodies, are complex proteins for which both safety and efficacy heavily depend on their three-dimensional structure. Detailed quality control of this aspect is both necessary and laborious. Aptamers are an emerging and potent tool in the quality control of such molecules, because they were shown to detect differences in the three-dimensional structure of a protein. In this study we aimed to generate DNA aptamers specific for a particular denaturation state of the anti-CD20 antibody rituximab. Utilizing the magnetic bead-based method of aptamer generation termed FluMag-SELEX, we managed to generate six aptamers against rituximab denatured at 80°C. Using bioinformatics tools, we predicted the aptamer structures, which we then further analysed using CD spectroscopy. The results suggested that five aptamers exhibit B-DNA-helix characteristics, while one aptamer forms G-quadruplex structures. Optimal aptamer working concentrations were titrated, and rituximab was treated at varying temperatures and time spans and then tested in an ELASA. All six aptamers were able to detect rituximab incubated at 80°C, but did not recognize the antibody in its native state. Incubating rituximab at 70°C for a longer duration also allowed the aptamers to bind, albeit to a lesser degree, suggesting that a comparable modification can be caused in rituximab at 70°C by increasing the time of exposure. Furthermore, subjecting rituximab to physical and UV stresses did not lead to the aptamers recognizing the protein, suggesting high specificity for epitopes established upon treatment at 80°C. All aptamers were also able to detect the IgG1 antibody adalimumab after it was incubated at 80°C, suggesting the antibody shares a binding motif with rituximab. With this study we could show that it is possible to generate aptamers that exclusively recognize a protein in its denatured conformation, but not in its native state. It is possible that in the future, these insights will prove to be valuable for use in the quality control processes of biopharmaceuticals.
|Publikationsstatus||Veröffentlicht - 2019|
Systematik der Wissenschaftszweige 2012
- 106 Biologie