Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Yu, Lei, Li, Yonghong, Tao, Lei, Jia, Chuncui, Yao, Wenrong, Rao, Chunming, Wang, Junzhi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479496/
https://www.ncbi.nlm.nih.gov/pubmed/30925680
http://dx.doi.org/10.3390/molecules24071210
_version_ 1783413358801715200
author Yu, Lei
Li, Yonghong
Tao, Lei
Jia, Chuncui
Yao, Wenrong
Rao, Chunming
Wang, Junzhi
author_facet Yu, Lei
Li, Yonghong
Tao, Lei
Jia, Chuncui
Yao, Wenrong
Rao, Chunming
Wang, Junzhi
author_sort Yu, Lei
collection PubMed
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.
format Online
Article
Text
id pubmed-6479496
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-64794962019-04-30 Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE Yu, Lei Li, Yonghong Tao, Lei Jia, Chuncui Yao, Wenrong Rao, Chunming Wang, Junzhi Molecules Article 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. MDPI 2019-03-28 /pmc/articles/PMC6479496/ /pubmed/30925680 http://dx.doi.org/10.3390/molecules24071210 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yu, Lei
Li, Yonghong
Tao, Lei
Jia, Chuncui
Yao, Wenrong
Rao, Chunming
Wang, Junzhi
Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479496/
https://www.ncbi.nlm.nih.gov/pubmed/30925680
http://dx.doi.org/10.3390/molecules24071210
work_keys_str_mv AT yulei identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT liyonghong identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT taolei identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT jiachuncui identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT yaowenrong identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT raochunming identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage
AT wangjunzhi identificationofarecombinanthumaninterleukin12rhil12fragmentinnonreducedsdspage