BM Chemiluminescence ELISA Substrate (Roche, Mannheim, Germany) was used and luminescence was measured with the Infinite M200 pro plate reader attenuation setting automatic mode (Tecan Group Ltd, M?nnedorf, Switzerland). Binding affinity studies using surface acoustic wave (SAW) technology The sam?5BLUE Purmorphamine biosensor instrument (NanoTemper Technologies, Munich, Germany) was used to determine the binding affinities of the different aptamers. 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 those 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. Introduction Biologics or biopharmaceuticals are a new generation of medicines produced by living organisms like bacteria, yeast, or mammalian cells1,2. Unlike small, chemically synthesised drugs, biologics are usually large recombinant proteins which are more difficult and cost-intensive to develop and produce. Biologics are typically guarded through patents; recent expirations of patent terms also allowed expansion in the field of biosimilars3,4. Biosimilars (or follow-on biologics in the United States) are defined as biological products highly similar to already approved biological medicines (reference medicine). In specific, those biosimilars do not show any clinically meaningful differences in terms of safety, purity, and efficacy from the reference product termed originator5,6. At the amino acid sequence level, biosimilars are designed to be identical to the originator. However, proposed biosimilars and originators may? still differ at the level of post-translational modifications due to differences in the highly complex production process. Such differences can potentially impact the safety, efficacy, and stability of pharmaceutical products. Therefore, detailed characterisation of the three-dimensional structure, post-translational modifications, and the aggregation behaviour of the protein is crucial to demonstrate similarity between the biosimilar and its reference product7C9. 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. This can however be restricted by the availability of appropriate antibody panels and also typically involves animal experiments for initial antibody generation10C12. An alternative approach to overcome these limitations is the application of aptamers. Aptamers, which are single-stranded DNA or RNA oligonucleotides with a specific three-dimensional structure, are typically obtained using the Purmorphamine selection process termed systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are able to bind various targets, such as proteins, small molecules, glycoproteins or even cells13C15. As they present a defined fold which can recognise a target with high affinity and specificity, they can be used as surrogate antibodies16C18. Unlike antibodies, aptamers can also be generated for targets that do not elicit immune responses as well as for toxic targets. A study from Zichel selection process SELEX. Six DNA aptamers reactive with rituximab were identified using ELASA. Binding affinities in the nanomolar range were determined and structural analyses revealed B-DNA helices and quadruplex structures. Robustness of the test assays was verified and specific binding mainly to the Fab fragment of rituximab was revealed. Selected aptamers were able to detect structural changes of thermally or UV light stressed rituximab. Analysis Mouse monoclonal to p53 of different rituximab biosimilar candidates revealed a high similarity between the products, while one aptamer was able to reveal a structural difference between the originator and a?proposed?copy product. Results selection of rituximab specific DNA aptamers 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. selection was performed by eight recurring incubations of rituximab-coated protein A magnetic beads using the folded single stranded oligonucleotides (Fig.?1a). Stringency of the SELEX process was increased in the last selection rounds by decreasing the amount of DNA incubated with the beads, increasing the number of washing steps and decreasing the number of PCR cycles. Additionally, a negative selection round was carried out with uncoated protein A magnetic beads before the last cycle. After cloning the DNA fragments of SELEX cycle eight into a cloning vector, plasmids from 50 clones were obtained and sequenced. Analysis of the 40 nucleotide Purmorphamine random part revealed fifteen different rituximab aptamer (RA) sequences with occurrences ranging from 1C12 times (Supplementary Table?S1). Open in a separate window Figure 1 selection and binding of aptamers. (a) Schematic representation of the SELEX process used within this study. (b) Schematic illustration of the?enzyme linked apta-sorbent assay (ELASA) setup. (c) Folded biotinylated aptamers (RA1-RA9, c?=?500?nM) were tested for rituximab binding. Bound aptamers were detected by streptavidin-HRP, chemiluminescence.