{"title":"SARS-CoV-2和Omicron疫苗从配体筛选到纯化的广谱亲和层析","authors":"Jing Ma , Xiaoyan Dong , Yan Sun , Qinghong Shi","doi":"10.1016/j.chroma.2025.465685","DOIUrl":null,"url":null,"abstract":"<div><div>Emerging variants of SARS-CoV-2 pose great technological and regulatory challenges to vaccine manufacturing, especially in downstream processing. To address this dilemma, the development of broad-spectrum affinity chromatography for the purification of wild-type SARS-CoV-2 and its variants is crucial. We propose a comprehensive strategy to achieve this goal via the identification of high-affinity peptides by affinity selection of phage display and next-generation sequencing (NGS) and the evaluation of chromatographic performance. Two peptides targeting the angiotensin-converting enzyme 2 (ACE2)-binding motif on the receptor-binding domain (RBD), HFVKTPARWAWG (SP-HFV) and HYRTSHWHHLLG (SP-HYR), were obtained from the most abundant sequences of the enriched phage library. They exhibited nanomolar affinity for the RBD and trimeric spike protein (Trimer S), and had broad-spectrum affinity for all the RBDs from the variants. Molecular dynamics simulations revealed the different binding regions of SP-HFV and SP-HYR in the ACE2-binding motif and key residues contributing to binding. After SP-HYR was coupled onto agarose matrices, chromatographic results showed that the RBD and Trimer S from the wild-type and Omicron variant could be adsorbed at pH 6.0–6.5 and eluted by increasing the salt concentration, exhibiting broad-spectrum and mild-elution characteristics of affinity chromatography. Finally, the affinity chromatography was applied for the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields (84.5–93.0 %) and purities (81.3–98.0 %), and great resistance to harsh cleaning-in-place in 20 cycles. This work clearly demonstrated the commercial potential of broad-spectrum affinity chromatography for vaccine purification to address the rapid variation of pathogenic viruses.</div></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":"1743 ","pages":"Article 465685"},"PeriodicalIF":4.0000,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Broad-spectrum affinity chromatography of SARS-CoV-2 and Omicron vaccines from ligand screening to purification\",\"authors\":\"Jing Ma , Xiaoyan Dong , Yan Sun , Qinghong Shi\",\"doi\":\"10.1016/j.chroma.2025.465685\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Emerging variants of SARS-CoV-2 pose great technological and regulatory challenges to vaccine manufacturing, especially in downstream processing. To address this dilemma, the development of broad-spectrum affinity chromatography for the purification of wild-type SARS-CoV-2 and its variants is crucial. We propose a comprehensive strategy to achieve this goal via the identification of high-affinity peptides by affinity selection of phage display and next-generation sequencing (NGS) and the evaluation of chromatographic performance. Two peptides targeting the angiotensin-converting enzyme 2 (ACE2)-binding motif on the receptor-binding domain (RBD), HFVKTPARWAWG (SP-HFV) and HYRTSHWHHLLG (SP-HYR), were obtained from the most abundant sequences of the enriched phage library. They exhibited nanomolar affinity for the RBD and trimeric spike protein (Trimer S), and had broad-spectrum affinity for all the RBDs from the variants. Molecular dynamics simulations revealed the different binding regions of SP-HFV and SP-HYR in the ACE2-binding motif and key residues contributing to binding. After SP-HYR was coupled onto agarose matrices, chromatographic results showed that the RBD and Trimer S from the wild-type and Omicron variant could be adsorbed at pH 6.0–6.5 and eluted by increasing the salt concentration, exhibiting broad-spectrum and mild-elution characteristics of affinity chromatography. Finally, the affinity chromatography was applied for the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields (84.5–93.0 %) and purities (81.3–98.0 %), and great resistance to harsh cleaning-in-place in 20 cycles. This work clearly demonstrated the commercial potential of broad-spectrum affinity chromatography for vaccine purification to address the rapid variation of pathogenic viruses.</div></div>\",\"PeriodicalId\":347,\"journal\":{\"name\":\"Journal of Chromatography A\",\"volume\":\"1743 \",\"pages\":\"Article 465685\"},\"PeriodicalIF\":4.0000,\"publicationDate\":\"2025-01-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Chromatography A\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0021967325000342\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Chromatography A","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0021967325000342","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Broad-spectrum affinity chromatography of SARS-CoV-2 and Omicron vaccines from ligand screening to purification
Emerging variants of SARS-CoV-2 pose great technological and regulatory challenges to vaccine manufacturing, especially in downstream processing. To address this dilemma, the development of broad-spectrum affinity chromatography for the purification of wild-type SARS-CoV-2 and its variants is crucial. We propose a comprehensive strategy to achieve this goal via the identification of high-affinity peptides by affinity selection of phage display and next-generation sequencing (NGS) and the evaluation of chromatographic performance. Two peptides targeting the angiotensin-converting enzyme 2 (ACE2)-binding motif on the receptor-binding domain (RBD), HFVKTPARWAWG (SP-HFV) and HYRTSHWHHLLG (SP-HYR), were obtained from the most abundant sequences of the enriched phage library. They exhibited nanomolar affinity for the RBD and trimeric spike protein (Trimer S), and had broad-spectrum affinity for all the RBDs from the variants. Molecular dynamics simulations revealed the different binding regions of SP-HFV and SP-HYR in the ACE2-binding motif and key residues contributing to binding. After SP-HYR was coupled onto agarose matrices, chromatographic results showed that the RBD and Trimer S from the wild-type and Omicron variant could be adsorbed at pH 6.0–6.5 and eluted by increasing the salt concentration, exhibiting broad-spectrum and mild-elution characteristics of affinity chromatography. Finally, the affinity chromatography was applied for the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields (84.5–93.0 %) and purities (81.3–98.0 %), and great resistance to harsh cleaning-in-place in 20 cycles. This work clearly demonstrated the commercial potential of broad-spectrum affinity chromatography for vaccine purification to address the rapid variation of pathogenic viruses.
期刊介绍:
The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.