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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
Source PublicationPROCESS BIOCHEMISTRY
ISSN1359-5113
Volume53Pages:216-223
Abstract

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.

KeywordDeamidation Tumor Necrosis Factor-alpha Lc-ms/ms Polyol Purification
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Technology
DOI10.1016/j.procbio.2016.11.011
Indexed BySCI
Language英语
WOS KeywordPurification ; Antibodies ; Proteins ; Aspartyl ; Antigen
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering, Chemical
Funding OrganizationNational 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 IDWOS:000394403400026
Citation statistics
Cited Times:1[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/21989
Collection生化工程国家重点实验室
Affiliation1.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
Recommended Citation
GB/T 7714
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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