Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.
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 metho...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3288073?pdf=render |
| _version_ | 1828873935457878016 |
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| author | Ran Zichel Wanida Chearwae Gouri Shankar Pandey Basil Golding Zuben E Sauna |
| author_facet | Ran Zichel Wanida Chearwae Gouri Shankar Pandey Basil Golding Zuben E Sauna |
| author_sort | Ran Zichel |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T07:26:42Z |
| format | Article |
| id | doaj.art-9b26de341a5946ec92d06426ff1f09fd |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T07:26:42Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-9b26de341a5946ec92d06426ff1f09fd2022-12-21T23:55:17ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0172e3194810.1371/journal.pone.0031948Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.Ran ZichelWanida ChearwaeGouri Shankar PandeyBasil GoldingZuben E SaunaTherapeutic 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.http://europepmc.org/articles/PMC3288073?pdf=render |
| spellingShingle | Ran Zichel Wanida Chearwae Gouri Shankar Pandey Basil Golding Zuben E Sauna Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. PLoS ONE |
| title | Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. |
| title_full | Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. |
| title_fullStr | Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. |
| title_full_unstemmed | Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. |
| title_short | Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins. |
| title_sort | aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins |
| url | http://europepmc.org/articles/PMC3288073?pdf=render |
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