目的 建立人源化抗血管内皮生长因子(vascular endothelial growth factor, VEGF)单克隆抗体的质控方法。方法 采用还原/非还原型毛细管凝胶电泳(CE-SDS)测定人源化抗VEGF单抗的纯度;在0.5和25.0 mg·mL-1浓度条件下,采用分子排阻高效液相色谱(SE-HPLC)测定制品中单体和聚合物含量;毛细管等电聚焦电泳(cIEF)测定其等电点;阳离子交换高效色谱(CEX-HPLC)测定电荷异构体含量;反相高效色谱(RP-HPLC)和LC-MS检测Lys-C酶切后制品肽图;人血管内皮细胞(human umbilical vein endothelial cells,HuVEC)增殖抑制试验测定其生物学活性;其余项目按照2015年版《中国药典》要求进行。结果 人源化抗VEGF单抗原液和成品的还原型CE-SDS的纯度分别(97.77±0.25)%和(97.43±0.57)%,非还原型CE-SDS纯度分别为(98.97±0.15)%和(98.73±0.06)%;0.5 mg·mL-1浓度下,原液和成品的单体含量分别为(98.07±0.55)%和(98.20±0.52)%;25.0 mg·mL-1浓度下,原液和成品的聚合物含量分别为(2.00±0.53)%和(1.93±0.55)%;CEX-HPLC检测原液和成品的酸性区含量分别为(18.33±0.64)%和(18.60±0.44)%,主峰含量分别为(69.03±0.80)%和(69.20±0.44)%,碱性区含量分别为(12.70±1.37)%和(12.20±0.87)%;cIEF和LC-MS测定原液和成品的等电点和肽图均与参比品一致;HUVEC增殖抑制法测定原液和成品的生物学活性分别为参比品的(107±6)%和(100±10)%。其他各项指标均符合《中国药典》要求及其他相关要求。结论 初步建立了人源化抗VEGF单克隆抗体的质控方法,可用于该制品的常规质量控制。
Abstract
OBJECTIVE To develop methods for quality control of humanized monoclonal antiboy (McAb) against vascular endothelial growth factor (VEGF).METHODS The purity of humanized anti-VEGF McAb was determined by reduced and non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS), while the contents of monomer and polymer were determined by size-exclusive high performance liqud chromatography (SE-HPLC) at the concentration of 0.5 and 25.0 mg·mL-1, respectively. Isoelectric point was determined by isoelectric focusing capillary electrophoresis (cIEF), and the content of charge isomers was controled by cation exchange HPLC(CEX-HPLC). For identity, Lys-C digested peptide map was analyzed by RP-HPLC and LC-MS. The biological potency of anti-VEGF McAb was determined with a human umbilical vein endothelial cells (HUVEC) proliferation inhibitory test. And other quality indexes required by the Ch.P and other relevant requirements were also investigated. RESULTS The purities of drug substance (DS) and products (DP) determined by reduced CE-SDS were (97.77±0.25)% and (97.43±0.57)%, respectively. The purities of DS and DP determined by non-reduced CE-SDS were (98.97±0.15)% and (98.73±0.06)%, respectively. In the SE-HPLC analysis, when the concentration of McAb was 0.5 mg·mL-1, the monomer contents of DS and DP were (98.07±0.55)% and (98.20±0.52)%, while the contents of polymer in DS and DP were (2.00±0.53)% and (1.93±0.55)% at the concentration of 25.0 mg·mL-1. In the CEX-HPLC analysis, the contents of the acidic, main, and basic components of DS and DP were (18.33±0.64)% and (18.60±0.44)%, (69.03±0.80)% and (69.20±0.44)%, (12.70±1.37)% and (12.20±0.87)%, respectively. In the cIEF and LC-MS tests, the DS and DP showed consistency in isoelectric point and peptide map with reference materials. The other quality indexes all met the requirements in Ch.P and other relevant requirements. CONCLUSION The mthods for quality control of McAb against VEGF is successfully developed, which can be used for the routine quality control of the product.
关键词
人血管内皮生长因子 /
人源化抗体 /
质量控制
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Key words
vascular endothelial growth factor /
humanized antibody /
quality control
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中图分类号：
R917
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参考文献
[1] FOLKMAN J. Tumor angiogenesis: therapeutic implications.[J]. N Engl J Med, 1971, 285(21):1182-1186.
[2] ALAM D S, YUNUS M,EL ARIFEEN S, et al. Zinc treatment for 5 or 10 days is equally efficacious in preventing diarrhea in the subsequent 3 months among Bangladeshi children[J]. J Nutr, 2010, 141(2):312-315.
[3] KERBEL R S. Tumor angiogenesis[J]. N Engl J Med, 2008, 358(19):2039-2049.
[4] MARTINY-BARON G, MARME D. VEGF-mediated tumour angiogenesis: a new target for cancer therapy[J]. Curr Opin Biotechnol, 1995, 6(6):675-680.
[5] SEMENZA G L. A new weapon for attacking tumor blood vessels[J]. N Engl J Med, 2008, 358(19):2066-2067.
[6] KEATING G M, SANTORO A. Sorafenib: a review of its use in advanced hepatocellular carcinoma[J]. Drugs, 2009, 69(2):223-240.
[7] GAN H K, SERUGA B, KNOX J J. Sunitinib in solid tumors[J]. Expert Opin Investig Drugs, 2009, 18(6):821-834.
[8] HURWITZ H. Integrating the anti-VEGF-A humanized monoclonal antibody bevacizumab with chemotherapy in advanced colorectal cancer[J]. Clin Colorectal Cancer, 2004,4(suppl 2):62-68.
[9] JEFFERIS R. Glycosylation of recombinant antibody therapeutics[J]. Biotechnol Prog, 2005, 21(1):11-16.
[10] NIMMERJAHN F, RAVETCH J V. Fcgamma receptors as regulators of immune responses[J]. Nat Rev Immunol, 2008, 8(1):34-47.
[11] BOEGGEMAN E, RAMAKRISHNAN B, PASEK M, et al. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection[J]. Bioconjug Chem, 2009, 20(6):1228-1236.
[12] HUTTERER K M, HONG R W, LULL J, et al. Monoclonal antibody disulfide reduction during manufacturing: untangling process effects from product effects[J]. MAbs, 2013, 5(4):608-613.
[13] RUSTANDI R R, WANG Y. Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress[J]. Electrophoresis, 2011, 32(21):3078-3084.
[14] PUROHIT V S, MIDDAUGH C R, BALASUBRAMANIAN S V. Influence of aggregation on immunogenicity of recombinant human Factor VIII in hemophilia A mice[J]. J Pharm Sci, 2006, 95(2):358-371.
[15] ROSENBERG A S. Effects of protein aggregates: an immunologic perspective[J]. Aaps J, 2006, 8(3):501-507.
[16] SHIRE S J, SHAHROKH Z, LIU J. Challenges in the development of high protein concentration formulations[J]. J Pharm Sci, 2004, 93(6):1390-1402.
[17] PHILO J S. A critical review of methods for size characterization of non-particulate protein aggregates[J]. Curr Pharm Biotechnol, 2009, 10(4):359-372.
[18] ZHANG A, SINGH S K, SHIRTS M R, et al. Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze-thaw stresses revealed by hydrogen exchange[J]. Pharm Res, 2012, 29(1):236-250.
[19] KHAWLI L A, GOSWAMI S, HUTCHINSON R, et al. Charge variants in IgG1: isolation, characterization, in vitro binding properties and pharmacokinetics in rats[J]. MAbs, 2010, 2(6):613-624.
[20] SHARIFI J, KHAWLI L A, HORNICK J L, et al. Improving monoclonal antibody pharmacokinetics via chemical modification[J]. Q J Nucl Med, 1998, 42(4):242-249.
[21] IGAWA T, TSUNODA H, TACHIBANA T, et al. Reduced elimination of IgG antibodies by engineering the variable region[J]. Protein Eng Des Sel, 2010, 23(5):385-392.
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脚注
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基金
国家科技重大专项重大新药创制资助项目(2014ZX09304311-001)
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