Abstract
Detailed analysis of biopharmaceuticals is crucial for safety, efficacy and stability. Aptamers, which are folded, single-stranded oligonucleotides, can be used as surrogate antibodies to detect subtle conformational changes. We aimed to generate and assess DNA aptamers against the therapeutic anti-CD20 antibody rituximab. Six rituximab-specific aptamers with Kd = 354–887 nM were obtained using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamer folds were analysed by online prediction tools and circular dichroism spectroscopy suggesting quadruplex structures for two aptamers while others present B-DNA helices. Aptamer binding and robustness with respect to minor differences in buffer composition or aptamer folding were verified in the enzyme-linked apta-sorbent assay. Five aptamers showed exclusive specificity to the Fab-fragment of rituximab while one aptamer revealed a broader recognition pattern to other monoclonal antibodies. Structural differences upon incubation at 40 °C for 72 h or UV exposure of rituximab were uncovered by four aptamers. High similarity between rituximab originator and biosimilar lots was demonstrated. The most sensitive aptamer (RA2) detected signal changes for all lots of a copy product suggesting conformational differences. For the first time, a panel of rituximab-specific aptamers was generated allowing the assessment of conformational coherence during production, storage, and biosimilarity of different products.
Highlights
Biologics or biopharmaceuticals are a new generation of medicines produced by living organisms like bacteria, yeast, or mammalian cells[1,2]
Aptamers, which are single-stranded DNA or RNA oligonucleotides with a specific three-dimensional structure, are typically obtained using the in vitro selection process termed systematic evolution of ligands by exponential enrichment (SELEX)
A DNA-library consisting of 1015 different single-stranded oligonucleotides with a random part of 40 nucleotides in length was used for selection of aptamers against the therapeutic IgG1 antibody rituximab
Summary
Biologics or biopharmaceuticals are a new generation of medicines produced by living organisms like bacteria, yeast, or mammalian cells[1,2]. There are only few and rather laborious analytical methods available, like NMR or X-ray crystallography, that are able to detect subtle changes in the tertiary structure of proteins Another method to monitor potential differences is the use of monoclonal antibodies specific to the target biologic. One of the most important biologics in cancer treatment, is a chimeric monoclonal anti-CD20 antibody produced in Chinese hamster ovary (CHO) cells[18,19]. It was the first monoclonal antibody (mAb) for the treatment of lymphoma approved by the Food and Drug Administration (FDA) in November 1997 and by the European Medicines Agency (EMA) in June 199819,20. Similarity between the biosimilar and the reference molecule in terms of safety, efficacy and quality needs to be demonstrated
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