Could Aptamers Be a New Feature in the Biosimilar Developer's Toolkit?

While biosimilar developers aim for their products to have identical amino acid sequences to their reference products, proposed biosimilars may still differ because of post-translational modifications, so detailed characterization of the 3-dimensional structure, post-translational modifications, and aggregation behavior of the protein is critical.

One emerging method to monitor potential differences is to apply aptamers: single-stranded DNA or RNA oligonucleotides. Aptamers can bind to various targets, and because they have a defined fold that can recognize a target with high affinity, they can be used as surrogate antibodies. A recent study reports on the effort to develop a panel of rituximab-specific aptamers that allow for the assessment of biosimilarity among rituximab products.

The study’s authors used the in vitro selection process SELEX to select and verify DNA aptamers against rituximab. They found 6 DNA aptamers to be reactive with rituximab, and they found specific binding, mainly to the Fab fragment of rituximab.

To investigate the specificity of the aptamers to rituximab, Fc fragments and other biologic drugs were tested. The investigators tested a glycosylated Fc fragment of rituximab, and a recombinant nonglycosylated Fc/2 obtained from E. coli. They also tested adalimumab, bevacizumab, and etanercept. Five of the 6 aptamers exclusively recognized full-length rituximab, pointing to high specificity.

The selected aptamers could also detect structural changes of thermally or UV light—stressed rituximab. When the investigators stored rituximab at different temperatures or exposed it to mechanical or UV light stress, 4 of the 6 aptamers could detect differences in the rituximab.

The investigators also used aptamers to investigate similarity among batches of EU-licensed reference rituximab (MabThera), a biosimilar rituximab (the European Medicines Agency-approved GP2013), and a “copy biologic” (Reditux, approved in India, but not approved in highly regulated territories). Five of the aptamers showed no statistically significant differences among the products, but 1 aptamer was able to reveal a difference between the copy biologic and the reference.

The paper’s authors write that aptamers, which are cost-efficient, could be implemented as an additional analytical method for developing and approving biosimilars that could address needs unmet by circular dichroism, Fourier transform infrared spectroscopy, or mass spectrometry.

“Based on our results, we suggest including the aptamer technology as orthogonal analytical approach in the portfolio of analytical techniques for characterization of biologics,” write the authors.

Reference

Wildner S, Huber S, Regl C, Huber CF, Lohrig U, Gadermaier G. Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab. Sci Rep. 2019;9: 1111. doi: 10.1038/s41598-018-37624-1.