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Open AccessArticle

Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE

National Institutes for Food and Drug Control, Beijing 100050, China
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Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editors: Angela R. Piergiovanni and José Manuel Herrero-Martínez
Molecules 2019, 24(7), 1210; https://doi.org/10.3390/molecules24071210
Received: 18 February 2019 / Revised: 21 March 2019 / Accepted: 23 March 2019 / Published: 28 March 2019
(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)
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Abstract

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. View Full-Text
Keywords: rhIL-12; purity; SDS-PAGE; SEC-HPLC; fragment; non-covalent binding rhIL-12; purity; SDS-PAGE; SEC-HPLC; fragment; non-covalent binding
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Yu, L.; Li, Y.; Tao, L.; Jia, C.; Yao, W.; Rao, C.; Wang, J. Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE. Molecules 2019, 24, 1210.

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