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.
Highlights
Interleukin-12 (IL-12) is a key inflammatory cytokine that critically influences Th1/Tc1-T cell responses at the time of an initial antigen encounter [1,2]
Generally known as protein multimers, were detected in both assays, but the relative percentage contents in size-exclusion chromatography (SEC)-HPLC were significantly higher than those in SDS-PAGE, which may be caused by the different running system—native for SEC-HPLC and denatured for SDS-PAGE
The denaturation led most of the non-covalent multimers to be depolymerized, so the multimers in SDS-PAGE were generally significantly lower than in SEC-HPLC
Summary
Interleukin-12 (IL-12) is a key inflammatory cytokine that critically influences Th1/Tc1-T cell responses at the time of an initial antigen encounter [1,2]. During the past two decades, IL-12 has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies [5,6,7]. IL-12 has multiple biological functions, though most importantly, it bridges early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity. Pharmaceutical companies have invested in the research and development of recombinant human IL-12 (rhIL-12) [5,10,11]. One such company in China has obtained a national first class clinical approval for rhIL-12 injection. Aside from this, some IL-12 fusion proteins were developed to minimize adverse effects, such as huBC1-IL-12, F8-IL-12 and IL-12-SS1
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