Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE
During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In o...
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| Format: | Article |
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MDPI AG
2019-03-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/24/7/1210 |
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| author | Lei Yu Yonghong Li Lei Tao Chuncui Jia Wenrong Yao Chunming Rao Junzhi Wang |
| author_facet | Lei Yu Yonghong Li Lei Tao Chuncui Jia Wenrong Yao Chunming Rao Junzhi Wang |
| author_sort | Lei Yu |
| collection | DOAJ |
| description | During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261–306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12. |
| first_indexed | 2024-12-23T20:20:12Z |
| format | Article |
| id | doaj.art-02488b2a864b4706ad993da0432859f8 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-23T20:20:12Z |
| publishDate | 2019-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-02488b2a864b4706ad993da0432859f82022-12-21T17:32:33ZengMDPI AGMolecules1420-30492019-03-01247121010.3390/molecules24071210molecules24071210Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGELei Yu0Yonghong Li1Lei Tao2Chuncui Jia3Wenrong Yao4Chunming Rao5Junzhi Wang6National Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaNational Institutes for Food and Drug Control, Beijing 100050, ChinaDuring the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261–306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.https://www.mdpi.com/1420-3049/24/7/1210rhIL-12puritySDS-PAGESEC-HPLCfragmentnon-covalent binding |
| spellingShingle | Lei Yu Yonghong Li Lei Tao Chuncui Jia Wenrong Yao Chunming Rao Junzhi Wang Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE Molecules rhIL-12 purity SDS-PAGE SEC-HPLC fragment non-covalent binding |
| title | Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE |
| title_full | Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE |
| title_fullStr | Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE |
| title_full_unstemmed | Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE |
| title_short | Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE |
| title_sort | identification of a recombinant human interleukin 12 rhil 12 fragment in non reduced sds page |
| topic | rhIL-12 purity SDS-PAGE SEC-HPLC fragment non-covalent binding |
| url | https://www.mdpi.com/1420-3049/24/7/1210 |
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