Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational\nchanges due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational\ndifferences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and\nstability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post\ntranslational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most\nproteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary\nstructure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and\nNMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies\ncan be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging.\nOther limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here\nwe provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize\nconformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to\ncharacterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been\nshown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We\ndemonstrate that a panel of aptamers (but not an antibody) can detect changes in the proteins even when specific activity\nis unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which\ncan be used in a routine drug-development and quality control setting. The technique can provide an early warning of\nstructural changes during the manufacturing process that could have consequential outcomes downstream.
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