Aptamers as a Sensitive Tool to Detect Subtle Modifications in Therapeutic Proteins

Abstract

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

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application

“…Recently, attention has turned toward aptamer production, due to the unique advantages of in vitro synthesis, its chemical component, high purity, and facilitation of modification, low molecular weight, high binding affinity, and equilibrium dissociation constant in the low nanomolar to high picomolar range, high stability and low immunogenicity. Also aptamers have emerged as a strong competitor of antibodies in analysis application, diagnostic biomaterial, therapeutic tools in the development of new drugs and targeted protein assay (Keefe et al, 2010;Mascini et al, 2012;McKeague and DeRosa, 2012;Nimjee et al, 2005;Ray et al, 2013;Zhu et al, 2012;Zichel et al, 2012). However, aptamers which are used in biomarker discovery respond to a critical need.…”

AptaTRACE Elucidates RNA Sequence-Structure Motifs from Selection Trends in HT-SELEX Experiments

“…Notably, under these assumptions, aptamers that contain only the sequence motif without the appropriate structural context are either not enriched at all, or enriched to a much lower degree. The second critical assumption we make is the existence of a multitude of sequence-structure binding motifs that either compete for the same binding site, or are binding to different surface regions of the target (Morris et al, 1998; Zichel et al, 2012). …”

“…This trend is in part attributable to the considerable diversity of possible targets, which include small organic molecules (Kim and Gu, 2013), transcription factors (Jolma et al, 2010) and other proteins or protein complexes (Berezhnoy et al, 2012), the surfaces of viruses (Binning et al, 2013), and entire cells [5,6,7] (Daniels et al, 2003; Morris et al, 1998; Shi et al, 2013). This broad range of targets makes aptamers suitable candidates for a variety of applications ranging from molecular biosensors (Zichel et al, 2012), to drug delivery systems (Upadhyay, 2015), and antibody replacement (FDA, 2004) to just name a few.…”

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

“…As this study demonstrates, fGmH aptamers are well-suited to conferring smartness to silver nanoparticles. It would not be a large logical leap to assume that such aptamers would also outperform aptamers of lower 2′-modification aptamers and antibody-based affinity molecules that have already been used to functionalize various biosensors, including those on atomic force microscopy, graphene, BLI, and SPR chips [37–40]. fGmH aptamers, specific to a variety of cancer biomarkers, could be used to decorate microfluidic platforms or biomimetic hydrogels for the capture and potential culture of circulating tumor cells (CTCs) derived from a plethora of cancer types, similar to that reported by using EpCAM antibodies [41–43].…”