|本期目录/Table of Contents|

[1]胡妍,罗利群,方树彬,等.FMU100抗体的超高效液相色谱法定量检测方法的建立[J].福建医科大学学报,2017,51(05):298-302.
 HU Yan,LUO Liqun,FANG Shubin,et al.Establishment of an UPLC Quantitative Method for FMU100 Antibody[J].Journal of Fujian Medical University,2017,51(05):298-302.
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FMU100抗体的超高效液相色谱法定量检测方法的建立(PDF)
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《福建医科大学学报》[ISSN:1672-4194/CN:35-1192/R]

卷:
第51卷
期数:
2017年05期
页码:
298-302
栏目:
论著
出版日期:
2017-10-30

文章信息/Info

Title:
Establishment of an UPLC Quantitative Method for FMU100 Antibody
文章编号:
16724194(2017)05029805
作者:
胡妍 罗利群 方树彬 温振国
作者单位: 福建医科大学 药学院免疫治疗研究所,福州350122
Author(s):
HU Yan LUO Liqun FANG Shubin WEN Zhenguo
Institute of Immune Therapy, Faculty of Pharmacy, Fujian Medical University, Fuzhou,350122,China
关键词:
色谱法高压液相 抗体
Keywords:
KEY WORDS:chromatography high pressure liquid antibodies
分类号:
R392; R446.9; R977.6; R979.5
DOI:
-
文献标志码:
A
摘要:
目的建立UPLCprotein A定量检测FMU100单克隆抗体的方法。方法使用Waters UPLC超高效液相色谱仪,Applied Biosystem的PA ImmunoDetection Sensor Cartridge(2.1×30 mm,20 μm)色谱柱,柱温30 ℃,检测波长280 nm,进样体积10 μL,以流动相A(10 mmol/L PBS含0.15 mmol/L NaCl, pH 7.2)流动相B(0.15 mmol/L NaCl, pH 2.6)进行梯度洗脱,流速0.5 mL/min。结果在0.034~2.200 mg/mL浓度,线性相关系数r2为0.999 9,浓度与峰面积间呈良好的线性关系;检测限为0.01 mg/mL,定量限为0.07 mg/mL;回收率为98.18%~99.16%;重复性RSD为0.1%~1.9 %;在8 h内,每个2 h测定发酵液中抗体的含量,FMU100抗体峰面积RSD为1.8%;在流动相A pH变化±0.2、流动相B NaCl比例变化±5%、柱温变化±5 ℃、检测波长变化±5 nm、流速相对值变化±20%时,FMU100抗体峰峰面积RSD为1.7%(n=12);检测方法符合系统适应性要求。结论建立的UPLCprotein A定量检测FMU100抗体的方法简单快速、方法可靠,符合方法学验证要求。
Abstract:
ABSTRACT:ObjectiveTo establish an UPLC|protein A quantitative method for FMU100 antibody and verify the methodology.MethodsAn Applied Biosystem PA ImmunoDetection Sensor Cartridge (2.1×30 mm,20 μm)was installed in UPLC with following parameters: the column temperature at 30 ℃, the detection wavelength of 280 nm and injection volume of 10 μL.The column was equilibrated by 10 mmol/L PBS (containing 0.15 mmol/L NaCl,pH 7.2) as mobile phase A for 4 minutes, then eluted by 0.15 mmo/L NaCl (pH 2.6) as mobile phase B for 4 minutes, reequilibrated by mobile phase A for 2 minutes at last, and the flow rate was 0.5 mL per min.ResultsThe increase peak areas were proportional to the concentration of FMU100 antibody in the range of 0.034~2.2 mg per mL, with linear correlation coefficient r2 0.999 9.The LOQ was 0.1 μg and DOQ was 0.7 μg.The recovery was 98.18%~99.10%.The RSD of repeatability was 0.1%~1.9 %.From the detection of antibodies content in fermented liquid in 8 h, at interval of 2 h, FMU100 antibodies peak area RSD was 1.8%.In the condition that pH varied ± 0.2 in mobile phase A, sodium chloride concentration in the mobile phase B varied ±5%, the column temperature varied ± 5 ℃,and the flow rate varied ± 20%,and the RSD of peak area was 1.7%.The test method met the requirement of system suitability with excellent specificity.ConclusionThe results showed that UPLC|protein A method is simple , quick, reliable, and meet the requirement of system suitability, therefore this method can be used for content determination and quality control of FMU100 antibody in the process of production.

参考文献/References:

[1]Vassiliki A,Boussiotis M D.Molecular and biochemical aspects of the PD1 checkpoint pathway[J]. New Engl J Med,2016,375(18):17671778.
[2]Brahmer J R,Drake C G,Wollner I,et al.Phase I study of singleagent antiprogrammed death1(MDX1106)in refractory solid tumors:safety,clinical activity,pharmacodynamics,and immunologic correlates[J]. J Clin Oncol,2010,28(19):31673175.
[3]Hirano F,Kaneko K,Tamura H,et al.Blockade of B7H1 and PD1 by Monoclonal Antibodies Potentiates Cancer Therapeutic Immunity[J]. Cancer Res,2005,65(3):10891096.
[4]王军志.生物技术药物研究开发和质量控制[M].北京:科学出版社,2007:8184.
[5]国家药典委员会.中华人民共和国药典[M].2015版 三部.北京:中国医药科技出版社,2015:通则5052.
[6]Gumustas M,Kurbanoglu S,Uslu B,et al.UPLC versus HPLC on drug analysis:advantageous, applications and their validation parameters[J]. Chromatographia,2013,76(21):13651427.
[7]Nováková L,Matysová L,Solich P.Advantages of application of UPLC in pharmaceutical analysis[J]. Talanta, 2006,68(3):908918.
[8]Forsgren A,Sjquist J.“Protein A” from S.Aureus I.Pseudoimmune reaction with human γglobulin[J]. J Immunol,1966,97(6):822827.
[9]Endresen C,Heggeness M,Grov A.Tryptic fragments of Fc from normal human IgG and their interaction with staphylococcal protein A[J]. Scand J Immunol,1974,3(3):261267.
[10]Hjelm H,Hjelm K,Sjquist J.Protein A from Staphylococcus aureus.Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins[J]. Febs Letters,1972,28(1):7376.
[11]国家药典委员会.中华人民共和国药典[M].2015版 三部.北京:中国医药科技出版社,2015:通则2325.
[12]王军志.生物技术药物研究开发和质量控制[M].北京:科学出版社,2007:119124.
[13]国家药典委员会.中华人民共和国药典[M].2015版 四部.北京:中国医药科技出版社,2015:374377.

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备注/Memo

备注/Memo:
基金项目: 福建省自然科学基金(2016J01154) 作者单位: 福建医科大学 药学院免疫治疗研究所,福州350122 作者简介: 胡妍,女,助理实验师,医学硕士 通讯作者: 温振国. Email: wenzhenguo@fjmu.edu.cn
更新日期/Last Update: 2017-10-30