检测重组人源抗人肿瘤坏死因子单克隆抗体甲氨蝶呤残留量的反相高效液相色谱法的建立及验证
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摘要
目的建立重组人源抗人肿瘤坏死因子单克隆抗体中甲氨蝶呤(methotrexate,MTX)残留量检测甲反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)法,并进行验证。方法样品经饱和硫酸铵法沉淀蛋白,取上清液进样检测。色谱柱为Waters/ACQUITY UPLC BEH C18(1. 7μm,2. 1 mm×100 mm),流动相为乙腈-甲醇-0. 054 mol/L磷酸二氢钾缓冲液(体积比6∶2∶92),pH 6. 5,检测波长302 nm,柱温:30℃,流速:0. 25 mL/min,进样体积:10μL,洗脱时间:15 min。同时验证方法的线性、特异性、准确度、定量限、精密性及耐用性。结果该方法特异性良好;MTX浓度在4～200 ng/mL范围内线性良好,R2> 0. 999;检测限为4 ng/mL;MTX浓度为20. 0、50. 0、100. 0 ng/mL 3个加标样品的回收率分别为102. 83%、100. 33%、99. 23%,3次重复检测结果RSD分别为1. 56%、1. 50%、0. 42%,两位实验员6次检测结果的RSD分别为2. 46%、1. 27%、1. 49%;MTX浓度为50. 0 ng/mL的加标样品在流动相p H 6. 3、6. 5、6. 7条件下测定结果的RSD为2. 02%。结论本实验建立的方法具有良好的特异性、精密性及准确度,且操作简便,可用于重组蛋白药物研发及生产过程中残留MTX的监测。
Objective To develop and verify a reversed-phase high performance liquid chromatography(RP-HPLC) for determination of residual methotrexate(MTX)content in recombinant monoclonal antibody against human tumor necrosis factor(TNF). Methods The protein in test samples was precipitated with saturated ammonium sulfate,while the supernatant was subjected to HPLC on a Waters/ACQUITY UPLC BEH C18 column(1. 7 μm,2. 1 mm × 100 mm)at30 ℃,using a mobile phase consisting of acetonitrile,methanol and 0. 054 mol/L potassium dihydrogen phosphate buffer(pH6. 5)at a ratio of 6 ∶ 2 ∶ 92 and a flow rate of 0. 25 mL/min. The wavelength for detection was 302 nm,while the volume of samples for loading was 10 μL,and the time for elution was 15 min. The developed method was verified for linearity, specificity, accuracy, quantitative limit,precision and durability. Results The developed method showed high specificity,of which the linear range was 4 ～ 200 ng/mL,with a R2 value of more than 0. 999. The detection limit of the method was 4 ng/mL. The spike recovery rates of MTX at concentrations of 20. 0,50. 0 and 100. 0 ng/mL were 102. 83%,100. 33% and 99. 23%,of which the RSDs in three repeat tests were 1. 56%,1. 50% and 0. 42%,and those in six test by two operators were 2. 46%,1. 27% and 1. 49%,respectively. The RSD of test results of spike samples of MTX at a concentration of 50. 0 ng/mL at pH 6. 3,6. 5 and 6. 7 was 2. 02%. Conclusion The developed method showed high specificity,precision and accuracy,and was simple to handle,which was suitable for determination of residual MTX content during development and production of recombinant protein drugs.
Objective To develop and verify a reversed-phase high performance liquid chromatography(RP-HPLC) for determination of residual methotrexate(MTX)content in recombinant monoclonal antibody against human tumor necrosis factor(TNF). Methods The protein in test samples was precipitated with saturated ammonium sulfate,while the supernatant was subjected to HPLC on a Waters/ACQUITY UPLC BEH C18 column(1. 7 μm,2. 1 mm × 100 mm)at30 ℃,using a mobile phase consisting of acetonitrile,methanol and 0. 054 mol/L potassium dihydrogen phosphate buffer(pH6. 5)at a ratio of 6 ∶ 2 ∶ 92 and a flow rate of 0. 25 mL/min. The wavelength for detection was 302 nm,while the volume of samples for loading was 10 μL,and the time for elution was 15 min. The developed method was verified for linearity, specificity, accuracy, quantitative limit,precision and durability. Results The developed method showed high specificity,of which the linear range was 4 ～ 200 ng/mL,with a R2 value of more than 0. 999. The detection limit of the method was 4 ng/mL. The spike recovery rates of MTX at concentrations of 20. 0,50. 0 and 100. 0 ng/mL were 102. 83%,100. 33% and 99. 23%,of which the RSDs in three repeat tests were 1. 56%,1. 50% and 0. 42%,and those in six test by two operators were 2. 46%,1. 27% and 1. 49%,respectively. The RSD of test results of spike samples of MTX at a concentration of 50. 0 ng/mL at pH 6. 3,6. 5 and 6. 7 was 2. 02%. Conclusion The developed method showed high specificity,precision and accuracy,and was simple to handle,which was suitable for determination of residual MTX content during development and production of recombinant protein drugs.
引文
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[2] FANG S P,LOLLO C P,DERUNES C,et al. Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities:yeastolates,triton X-100 and methotrexate[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2011,879(30):3612-3619.
[3] LI H W,CHEN K M,WANG Z,et al. Genetic analysis of the clonal stability of Chinese hamster ovary cells for recombinant protein production[J]. Mol BioSyst,2016,12(1):102-109.
[4] HU X Q,HAO F,LI Z G,et al. Research progress on quality control of impurities or hazardous substances in drugs[J]. Drugs&Clinic,2014,29(9):953-964.(in Chinese)胡向青,郝福,李志刚,等.药物中杂质及有害物质控制限度的研究进展[J].现代药物与临床,2014,29(9):953-964.
[5] LI M,CHANG W H. Principles for specification establishment of biological products[J]. Chin J N Drugs,2017,26(16):1887-1893.(in Chinese)李敏,常卫红.生物制品质量标准研究与建立一般原则的探讨[J].中国新药杂志,2017,26(16):1887-1893.
[6] ZHANG Y Y,XU K K. Comparison of three different methods in determination of methotrexate in plasma[J]. Anhui Med Pharm J,2015,18(2):357-360.(in Chinese)张媛媛,徐康康.三种检测方法测定甲氨蝶呤血药浓度的比较分析[J].安徽医药,2015,18(2):357-360.
[7] SHI X,YAO Q,WU S J. Serum concentration monitoring and significance of methotrexate in patients with osteosarcoma undergoing chemotherapy[J]. Jiangsu Med J,2010,36(8):869-871.(in Chinese)施鑫,姚强,吴苏稼.甲氨蝶呤化疗的血药浓度监测及意义[J].江苏医药,2010,36(8):869-871.
[8] WU D Y,LIU D,DONG M. Literature analysis for the current status of methotrexate plasma concentration monitoring in China[J]. China Pharmacy,2016,27(33):4665-4667.(in Chinese)吴东媛,刘铎,董梅.我国甲氨蝶呤血药浓度监测文献分析[J].中国药房,2016,27(33):4665-4667.
[9] Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Repubic of China(VolⅡ)[S]. Beijing:China Med Sci Press,2015:208.(in Chinese)国家药典委员会.中华人民共和国药典(二部)[S].北京:中国医药科技出版社,2015:208.
[10] CHENG B,LIN N M,FANG L. RP-HPLC determination of methotrexate in human plasma[J]. Chin J Pharm Anal,2006,26(5):598-601.(in Chinese)程斌,林能明,方罗. RP-HPLC测定人体血浆中甲氨蝶呤的浓度[J].药物分析杂志,2006,26(5):598-601.
[11] LIU Y,GE M J,ZHAI S D. Determination of methotrexate in human plasma by high performance liquid chromatography detector[J]. Chin J Clin Pharm,2016,32(18):1705-1708.(in Chinese)刘洋,戈梦佳,翟所迪.高效液相色谱法测定人血浆中甲氨蝶呤的浓度[J].中国临床药理学杂志,2016,32(18):1705-1708.
[12] HUANG M M,PENN L,BONGERS J,et al. Validation of a shielded-hydrophobic-phase high-performance liquid chromatography method for the determination of residual methotrexate in recombinant protein biopharmaceuticals[J]. J Chromatogr A,1998,828(1-2):303-309.