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人脐静脉内皮细胞增殖抑制法检测贝伐珠单抗活性的优化和应用
人脐静脉内皮细胞增殖抑制法检测贝伐珠单抗活性的优化和应用
张 峰,徐刚领,于传飞,王文波,陈 伟,刘春雨,王 兰,高 凯

中国食品药品检定研究院单克隆抗体产品室,北京 100050
Optimization and application of HUVEC proliferation inhibitory assay
(Division of Monoclonal antibody Products, National Institutes for Food and Drug Control, Beijing 100050, China)

摘要参考文献相关文章

起始页:2317

摘要:目的:优化人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)增殖抑制法,并用优化后方法比较6种贝伐珠单抗(bevacizumab)生物类似药(similar bio-therapeutic products,SBPs)候选药物与原研药的生物学活性。方法:对细胞接种量、VEGF165浓度和样品稀释浓度进行优化,提高方法精密性和准确性。使用优化后的方法,以贝伐珠单抗为参比品,测定6种贝伐珠SBPs的生物学活性,并与原研药进行比较。结果:优化后的HUVEC增殖抑制法,信噪比为1.5~1.8,板间变异度<20%,在70%~130%活性范围内有良好的准确性。使用优化后方法检测,质控样品的相对活性均值为102.00%,6种贝伐珠SBPs的相对活性分别为95.12%,88.81%,96.96%,103.00%,105.70%和102.40%。结论:通过优化,HUVEC增殖抑制法的精密性和准确性较经典方法有显著提高,适用于贝伐珠单抗及其SBPs的可比性评价。

关键词:血管内皮细胞生长因子;单抗;生物学活性;质量控制

通讯作者:高凯,男,研究员,主要从事治疗性单克隆抗体产品的质量研究工作。

基金项目:国家“重大新药创制”科技重大专项(2014ZX09304311-001)

作者简介:张峰,男,副主任技师,主要从事治疗性单克隆抗体产品的质量研究工作。

Abstract:Objective: To optimize human umbilical vein endothelial cell (HUVEC ) proliferation inhibitory assay, and compare the biological potency of 6 bevacizumab similar bio-therapeutic products (SBPs) with bevacizumab. Methods: Cell inoculum density, VEGF165 concentration and sample concentration were optimized to improve repeatability and accuracy of the method. Using optimized method, relative potencies of 6 bevacizumab SBPs were determined and compared with original drug. Results: After optimization, the signal/noise ratio was 1.5~1.8, and variation <20% within plates. The optimized assay had a good repeatability and accuracy within the potency range of 70%~130%. Using the optimized assay, the relative potency of product control was 102.00%. Relative potencies of 6 bevacizumab SBPs were determined as 95.12%,88.81%,96.96%,103.00%,105.70% and 102.40%. Conclusion: By optimization, the precision and accuracy of HUVEC proliferation inhibition assay are significantly elevated, which meets the requirements of potency comparison studies on bevacizumab and its SBPs.

Key words:vascular endothelial growth factor (VEGF); monoclonal antibody; biological potency; quality control



    [1] FOLKMAN J. Tumor anagiogenesis: therapeutic implications

    [J]. New Engl J Med, 1971, 285(21): 1182-1186.


    [2] CHOWDURY MW, SCARAMUZZI RJ, WHEELER-JONES CP, et al. The expression of angiogenic growth factors and their receptors in ovarian follicles throughoutt the estrous cycle in the in the ewe

    [J]. Theriogenology, 2010, 73(7):856-872.


    [3] TENG LS,JIN KT,HE KF, et al. Advances in combination of antiangiogenic agents targeting VEGF-binding and conventional chemotherapy and radiation for cancer treatment

    [J]. J Chin Med Assoc, 2010, 73(6):281-288.


    [4] KERBEL RS. Tumor angiogenesis

    [J]. N Engl J Med, 2008, 358(19):2039-2049.


    [5] MARTINY-BARON G, MARM D. VEGF mediated tumour angiogenesis: a new target for cancer therapy

    [J]. Curr Opin Biotechnol, 1995,6(6):675-680.


    [6] ELLIS LM, HICKLIN DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity

    [J].Nat Rev Cancer, 2008, 8(8):579-591.


    [7] HURWITZ H. Integrating the anti-VEGFA humanized monoclonal antibody bevacizumab with chemotherapy in advanced colorectal cancer

    [J]. Clin colorectal Cancer, 2004, 4(Suppl 2):S62-S68.


    [8] FERRARA N,HILLAN KJ,NOVOTNY W. Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy

    [J]. Biochem Biophys Res Commun, 2005, 333(2):328-335.


    [9] 陈薇, 张晶, 刘强, 等. 血管内皮生长因子全长抗体在毕赤酵母中的表达与活性鉴定

    [J]. 生物技术通讯, 2014, 25(4):492-496.


    [10] 石金平, 赵健. 抗人VEGF及OPN双特异性抗体的制备及生物学活性研究

    [D]. 上海:第二军医大学, 2009:43.


    [11] 范文红, 毕华, 饶春明, 等. 重组人源化兔抗血管内皮生长因子单克隆抗体的生物学活性检测方法的建立

    [J]. 中国生物制品学杂志, 2012, 25(3):414-417.


    [12] HAFIZAH AH, ZAITON Z, ZULKHAIRI A, et al. Piper sarmentosum as an antioxidant on oxidative stress in human umbilical vein endothelial cells induced by hydrogen peroxide

    [J]. J Zhejiang Univ Sci B, 2010, 11(5):357-365.


    [13] 徐蓉,徐金勇,刘玮. 骨髓间充质干细胞定向分化为血管内皮样细胞的实验研究

    [J]. 生物医学工程学杂志,2014,31(2):389-393.


    [14] TAKAHASHI H, SHIBUYA M. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions

    [J]. Clin Sci(Lond), 2005, 109(3):272-241.


    [15] TASHIRO S, TSUMOTO K, SANO E. Establishment of a microcarrier culture system with serial sub-cultivation for functionally active human endothelial cells

    [J]. J Biotechnol, 2012, 160(3-4):202-213.


    [16] HEISS M, HELLSTRM M, KALN M, et al. Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro

    [J]. FASEB J, 2015, 29(7):3076-3084.