DOE法优化筛选表达胰高血糖素样肽-1融合蛋白的CHO细胞用培养基
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摘要
目的运用实验设计(Design of Experiment,DOE)方法优化筛选表达胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)融合蛋白的CHO细胞用培养基,以提高细胞生长密度及目的蛋白表达量。方法采用Minitab软件对实验进行DOE优化筛选。考察不同基础培养基、流加培养基及其策略优化对CHO活细胞密度(viable cell density,VCD)与GLP-1融合蛋白表达的影响,确定最适培养基组合。结果从15种基础培养基中筛选出3组基础培养基,从5种流加培养基中筛出1种流加培养基,并在此基础上优化了相关流加补料策略。经最适培养基组合验证,试验组CHO细胞的VCD最高为(19. 90±0. 78)×10~6个/mL,对照组最高为(14. 30±0. 60)×10~6个/mL;试验组GLP-1融合蛋白表达最高为2. 51 g/L,对照组小于1. 51 g/L,目的蛋白表达量优化后较优化前提高了80%以上。结论优化后的培养基组合显著提高了CHO细胞生长密度及目的蛋白产量,DOE方法是一种短期快速提高蛋白表达的有效方法。
Objective To optimize the medium for expression of glucagon-like peptide-1(GLP-1 fusion protein)in Chinese hamster ovary(CHO)cells by Design of Experiment(DOE). Methods DOE experiments were performed with Minitab software to investigate the effects of various basal media and feed medium on the viable cell density of CHO cells and the expression of GLP-1 fusion protein,based on which the optimal combination of medium was screened. Results Three groups from fifteen kinds of basal medium as well as one kind from five kinds of feed medium were screened,based on which the relevant strategy for fed batch was modified. The highest viable cell density in test and control groups were(19. 90 ± 0. 78)× 10~6 and(14. 30 ± 0. 60)× 10~6 cells/m L,while highest expression levels of GLP-1 fusion protein were2. 51 and 1. 51 g/L,respectively. The expression level of target protein in medium after optimization increased by more than 80% as compared with that before optimization. Conclusion After optimization of the medium,both the cell growth density and protein expression level increased significantly,indicating that DOE was an effective method for rapidly increasing protein expression in short term.
Objective To optimize the medium for expression of glucagon-like peptide-1(GLP-1 fusion protein)in Chinese hamster ovary(CHO)cells by Design of Experiment(DOE). Methods DOE experiments were performed with Minitab software to investigate the effects of various basal media and feed medium on the viable cell density of CHO cells and the expression of GLP-1 fusion protein,based on which the optimal combination of medium was screened. Results Three groups from fifteen kinds of basal medium as well as one kind from five kinds of feed medium were screened,based on which the relevant strategy for fed batch was modified. The highest viable cell density in test and control groups were(19. 90 ± 0. 78)× 10~6 and(14. 30 ± 0. 60)× 10~6 cells/m L,while highest expression levels of GLP-1 fusion protein were2. 51 and 1. 51 g/L,respectively. The expression level of target protein in medium after optimization increased by more than 80% as compared with that before optimization. Conclusion After optimization of the medium,both the cell growth density and protein expression level increased significantly,indicating that DOE was an effective method for rapidly increasing protein expression in short term.
引文
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[20]李惠钰.技术领跑抗体产业“马拉松”[N].中国科学报,2013,8(7):第8版.
[2] ZHANG Z,ZHANG W,JUNG D Y,et al. TRPM2 Ca2+channel regulates energy balance and glucose metabolism[J]. Am J Physiol Endocrinol Metab,2012,302(7):E 807-E816.
[3] FREEMAN J S. Improving glucagon-like peptide-1 dynamics in patients with type 2 diabetes mellitus[J]. J Am Osteopath Assoc,2012,112(1 Suppl 1):S2-S6.
[4] HINNEN D A. Therapeutic options for the management of postprandial glucose in patients with type 2 diabetes on basal insulin[J]. Clin Diabetes,2015,33(4):175-180.
[5] KIM J Y,KIM Y G,LEE G M. CHO cells in biotechnology for production of recombinant proteins:current state and further potential[J]. Appl Microbiol Biotechnol,2012,93(3):917-930.
[6] DIETMAIR S,HODSON M P,QUEK L E,et al. Metabolite profiling of CHO cells with different growth characteristics[J].Biotechnol Bioeng,2012,109(6):1404-1414.
[7] ZHOU G Y,LIU B L,HAN Y Y. Design and analysis the orthogonal experiment with interaction of factors using minitab[J]. Educat Tech Forum,2017,9(44):275-276.(in Chinese)周国燕,刘宝林,韩颖颖. Minitab实现有交互作用的正交实验的设计与结果分析[J].教育教学论坛,2017,9(44):275-276.
[8] PENG A H. Application of Minitab software in orthogonal experimental design with duplicate test[J]. J Jimei Univ,2013,14(1):111-114.(in Chinese)彭爱红. Minitab软件在有重复试验的正交试验设计中的应用[J].集美大学学报,2013,14(1):111-114.
[9] RODRIGUES M E,COSTA A R,HENRIQUES M,et al. Advances and drawbacks of the adaptation to serum-free culture of CHO-K1cells for monoclonal antibody production[J]. Appl Biochem Biotechnol,2013,169(4):1279-1291.
[10] RODRIGUES M E,COSTA A R,HENRIQUES M,et al.Comparison of commercial serum-free media for CHO-K1 cell growth and monoclonal antibody production[J]. Int J Pharm,2012,437(1-2):303-305.
[11] COSTA A R,WITHERS J,RODRIUES M E,et al. The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells[J]. N Biotechnol,2013,30(5):563-572.
[12] LING W L,BAI Y,CHENG C,et al. Development and manufacturability assessment of chemically-defined medium for the production of protein therapeutics in CHO cells[J]. Biotechnol Prog,2015,31(5):1163-1171.
[13] ALTAMIRANO C,ILLANES A,BECERRA S,et al. Considerations on the lactate consumption by CHO cells in the presence of galactose[J]. J Biotechnol,2006,125(4):547-556.
[14] HONG J K,CHO S M,YOON S K. Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells[J]. Appl Microbiol Biotechnol,2010,88(4):869-876.
[15] ALTAMIRANO C,PAREDES C,CAIRO J J,et al. Improvement of CHO cell culture medium formulation:simultaneous substitution of glucose and glutamine[J]. Biotechnol Prog,2000,16(1):69-75.
[16] XING Z,KENTY B M,LI Z J,et al. Scale-up analysis for a CHO cell culture process in large-scale bioreactors[J].Biotechnol Bioeng,2009,103(4):733-746.
[17] BRüHLMANN D,SOKOLOV M,BUTTéA,et al. Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality[J]. Biotechnol Bioeng,2017,114(7):1448-1458.
[18] YUAN Z M,ZUO B,TAN S Q,et al. Experimental design and analysis of the optimal fermentation medium based on uniform design and support vector regression[J]. Chin J Proc Eng,2009,9(1):148-452.(in Chinese)袁哲明,左斌,谭泗桥,等.基于均匀设计与支持向量回归的发酵配方优化[J].过程工程学报,2009,9(1):148-152.
[19] LARA E F,LINK H,BOTZ D W. Evaluation of artificial neural networks for modeling and optimization of medium composition with a genetic algorithm[J]. Proc Biochem,2006,41(10):2200-2206.
[20]李惠钰.技术领跑抗体产业“马拉松”[N].中国科学报,2013,8(7):第8版.
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