title: Quality-control method for the determination of biological activity of engineered calcineurin subunit B.
abstract: The aim of this study was to establish a quality-control method for calcineurin subunit B (CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate (pNPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant (CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding pNPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05-50 μg mL(-1), with sensitivity of 50 μg mL(-1). Tested cytokines did not induce CNAΔ316 dephosphorylation of pNPP. The chosen CNAΔ316 concentration range did not affect activity determinations.
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, 100050, China.
authors.forename: Xinchang
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, 100050, China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.affiliation: Haikou Qili Pharmaceutical CO., LTD., Haikou, 570216, China.
authors.forename: Zongwen
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Haikou Qili Pharmaceutical CO., LTD., Haikou, 570216, China.
authors.forename: Yudong
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, China. weiq@bnu.edu.cn.
authors.forename: Qun
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, 100050, China. raocm@nicpbp.org.cn.
authors.forename: Chunming
authors.initials: C
authors.lastname: Rao
mesh_terms: D019703:Calcineurin / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D057230:Limit of Detection; D011786:Quality Control; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins; D019703:Calcineurin
keywords: biological activity; calcineurin subunit B; method establishment; method validation; quality control