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Identification, characterization, and stabilization of the deamidation degradation of recombinant human tumor necrosis factor-alpha
Yin, Shuang1,2; Zhang, Chun1,2; Li, Zenglan2; Wang, Qi2; Shi, Hong2; Yu, Rong1; Liu, Yongdong2; Su, Zhiguo2
2017-02-01
发表期刊PROCESS BIOCHEMISTRY
ISSN1359-5113
卷号53页码:216-223
摘要A kind of degradation characterized by an increase in overall negative charge in both native polyacrylamide gel electrophoresis analysis and high-performance strong anion exchange analysis was observed during the purification process of recombinant human tumor necrosis factor-alpha (TNF-alpha). Liquid chromatography coupled with tandem mass spectrometry was adopted to further analyze this degradation, and the result demonstrated that suspected deamidation occurred at N39 and N34 residues. To investigate the effects of these deamidation degradations on TNF-alpha, we substituted corresponding asparagine residues with aspartic acid residues. High-performance size-exclusion chromatography, circular dichroism, and fluorescence spectrometry analysis revealed that the advanced structures of TNF-alpha could not be obviously changed by these substitutions. Differential scanning calorimetry analysis indicated that deamidation led to decreased thermal stability, and two mutants (N34D, N34DN39D) both possessed two Tm. L929 cell cytotoxic activity implied that N39 residue deamidation caused only a minor bioactivity loss, whereas N34 residue deamidation led to a bioactivity loss of four orders of magnitude. To alleviate the degradation during the purification process, we screened nine excipients and found that glycerol could notably ameliorate this degradation and provide a compromise strategy for the recombinant human TNF-alpha protein during purification process and formulation development. (C) 2016 Published by Elsevier Ltd.
关键词Deamidation Tumor Necrosis Factor-alpha Lc-ms/ms Polyol Purification
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine ; Technology
DOI10.1016/j.procbio.2016.11.011
收录类别SCI
语种英语
关键词[WOS]PURIFICATION ; ANTIBODIES ; PROTEINS ; ASPARTYL ; ANTIGEN
WOS研究方向Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering
WOS类目Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering, Chemical
项目资助者National Natural Science Foundation of China(21576267) ; Beijing Natural Science Foundation(2162041) ; Novo Nordisk Chinese Academy of Sciences Research Fund(NNCAS-2014-02) ; Open Funding Project of the National Key Laboratory of Biochemical Engineering(2014KF-04)
WOS记录号WOS:000394403400026
引用统计
被引频次:1[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/21989
专题生化工程国家重点实验室
作者单位1.Sichuan Univ, West China Sch Pharm, Minist Educ, Key Lab Drug Targeting & Drug Delivery Syst, Chengdu 610041, Peoples R China
2.Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100080, Peoples R China
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Yin, Shuang,Zhang, Chun,Li, Zenglan,et al. Identification, characterization, and stabilization of the deamidation degradation of recombinant human tumor necrosis factor-alpha[J]. PROCESS BIOCHEMISTRY,2017,53:216-223.
APA Yin, Shuang.,Zhang, Chun.,Li, Zenglan.,Wang, Qi.,Shi, Hong.,...&Su, Zhiguo.(2017).Identification, characterization, and stabilization of the deamidation degradation of recombinant human tumor necrosis factor-alpha.PROCESS BIOCHEMISTRY,53,216-223.
MLA Yin, Shuang,et al."Identification, characterization, and stabilization of the deamidation degradation of recombinant human tumor necrosis factor-alpha".PROCESS BIOCHEMISTRY 53(2017):216-223.
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