超高效液相色谱法测定人血白蛋白分子大小分布方法的建立
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摘要
目的建立人血白蛋白(human serum albumin,HSA)产品分子大小分布(多聚体含量)的超高效液相色谱(ultra-performance liquid chromatography,UPLC)分析测定方法。方法采用Waters ACQUITYUPLC系统、Waters TUV紫外检测器、ACQUITY UPLC PROTEIN BEH SEC色谱柱,以磷酸盐缓冲液作为流动相、0.6 mL·min~(-1)流速、TUV紫外280 nm测定人血白蛋白分子大小分布。稀释制备不同质量浓度(0.5~20 mg·mL~(-1))的HSA样品分析不同进样浓度下其各组分百分含量变化、线性关系等,分子大小分布结果按照面积归一化法计算。在最佳进样浓度范围内,选择适宜浓度稀释人血白蛋白国家参考品并用UPLC测定进行方法学确认。用UPLC和HPLC方法扩展测定20批人血白蛋白样品,进行方法一致性比对和再确认。结果 UPLC测定人血白蛋白多聚体保留时间为1.469 min,二聚体保留时间为1.972 min,单体峰保留时间为2.267 min。二聚体与单体分离度为2.20,单体峰拖尾因子1.18。11个进样质量浓度条件下(0.5~20 mg·mL~(-1))人血白蛋白样品各组分包括多聚体、二聚体、单体峰的峰高及峰面积与进样浓度之间均呈良好线性关系,线性相关系数平方均大于0.997 0;不同浓度下多聚体峰面积百分比相对标准偏差RSD分别为0(n=3,10 mg·mL~(-1))和0.10%(n=3,12 mg·mL~(-1))和0.10%(n=3,16mg·mL~(-1));人血白蛋白样品在10~16 mg·mL~(-1)(上样总量100~160μg)内各组分峰面积百分比及与进样浓度的线性关系均保持相对稳定;HPLC测定人血白蛋白国家参考品多聚体峰面积百分含量为5.58%,UPLC平行测定该国家参考品多聚体含量均值为5.62%(n=10),两者一致且结果均符合国家参考品的质量控制范围;UPLC测定人血白蛋白国家参考品多聚体峰面积百分比RSD为0.40%(n=10);HPLC与UPLC方法平行测定的20批来自国内外7家企业的人血白蛋白样品(12mg·mL~(-1)下)多聚体峰面积百分含量结果证实2种方法无显著性差异(P>0.05),2种方法的相关系数为0.996 0(n=20)。结论 UPLC测定人血白蛋白产品的多聚体含量准确性和重复性良好,相比于传统HPLC方法新建UPLC方法大幅节省分析时间,更为准确、快速、简便,可实现高通量的人血白蛋白分子大小分布测定。
OBJECTIVE To establish a determination method of molecular-size distribution of human serum albumin( HSA) by ultra performance liquid chromatography( UPLC). METHODS An UPLC method was developed to specifically determine the polymers and other components in HSA on UPLC PROTEIN BEH SEC analysis column with Waters Alliance UPLC system and Waters UPLC TUV detector. The separation was performed using a mobile phase consisting of PBS at a flow rate of 0. 6 mL·min~(-1) and the UV detection wavelength was set at 280 nm. HSA samples were diluted to different concentrations( 0. 5-20 mg·mL~(-1)) to confirm the optimal concentration range of the injection. The change of component percentage and the linear relationship between HSA concentration and chromatographic peak height were confirmed and the molecular-size distribution was calculated by area normalization method.Within the optimum injection concentration range,the national control sample for HSA was diluted to 12 mg·mL~(-1) and tested by UPLC method and the methodology was confirmed. Twenty batches of HSA samples were determined by both UPLC and existing HPLC methods,and the samples were determined in parallel. The consistency of the methods was compared and the method was reconfirmed.RESULTS The UPLC retention time of HSA polymer was 1. 469 min,of dimer was 1. 972 min,and of monomer was 2. 267 min,respectively. The resolution of dimer and monomer was 2. 20 and the USP tailing of monomer peak was 1. 18 respectively. In the range of the injection concentrations,0. 5-20 mg·mL~(-1),there was linear relationship between the concentrations of the components of 11 HAS samples,including polymer,dimer and monomer peak,and the peak area%,peak height,peak area,and the squares of linear correlation coefficient were all greater than 0. 997 0. The components peak area percentage of HSA samples remained relatively stable within the concentration range of 10-16 mg·mL~(-1)( total injection amount of 100-160 μg). The RSDs of the percentage of polymers were 0. 00%( n = 3,10 mg·mL~(-1)),0. 10%( n = 3,12 mg·mL~(-1)),and 0. 10%( n = 3,16 mg·mL~(-1)),respectively. The UPLC method was used to determine the national control sample for human albumin of 12 mg·mL~(-1),and the mean value of peak area%was 5. 62%( n = 10). The results were consistent with those of the parallel determination by HPLC( 5. 58%),both of which were in accordance with the quality control range of the national standard for human albumin. The RSD of the percentage of the peak area of the polymers in national standard for human albumin by UPLC was 0. 40%( n = 10). The HPLC and UPLC methods were used to determine the polymer peak area percentage of 20 batches of HSA samples from 7 domestic and foreign enterprises at the concentration of 12 mg·mL~(-1). The correlation coefficient of the two methods was 0. 996 0( P > 0. 05) and there was no significant difference between the two methods( P > 0. 05). CONCLUSION Compared with the traditional HPLC method,the detection time of HSA SEC by the proposed UPLC method is shortened by at least 10 times,and the accuracy and repeatability of the determination are satisfactory. UPLC method can save much more analysis time,is simple and much faster,and can be used for high-throughput determination of molecularsize distribution of human serum albumin.
OBJECTIVE To establish a determination method of molecular-size distribution of human serum albumin( HSA) by ultra performance liquid chromatography( UPLC). METHODS An UPLC method was developed to specifically determine the polymers and other components in HSA on UPLC PROTEIN BEH SEC analysis column with Waters Alliance UPLC system and Waters UPLC TUV detector. The separation was performed using a mobile phase consisting of PBS at a flow rate of 0. 6 mL·min~(-1) and the UV detection wavelength was set at 280 nm. HSA samples were diluted to different concentrations( 0. 5-20 mg·mL~(-1)) to confirm the optimal concentration range of the injection. The change of component percentage and the linear relationship between HSA concentration and chromatographic peak height were confirmed and the molecular-size distribution was calculated by area normalization method.Within the optimum injection concentration range,the national control sample for HSA was diluted to 12 mg·mL~(-1) and tested by UPLC method and the methodology was confirmed. Twenty batches of HSA samples were determined by both UPLC and existing HPLC methods,and the samples were determined in parallel. The consistency of the methods was compared and the method was reconfirmed.RESULTS The UPLC retention time of HSA polymer was 1. 469 min,of dimer was 1. 972 min,and of monomer was 2. 267 min,respectively. The resolution of dimer and monomer was 2. 20 and the USP tailing of monomer peak was 1. 18 respectively. In the range of the injection concentrations,0. 5-20 mg·mL~(-1),there was linear relationship between the concentrations of the components of 11 HAS samples,including polymer,dimer and monomer peak,and the peak area%,peak height,peak area,and the squares of linear correlation coefficient were all greater than 0. 997 0. The components peak area percentage of HSA samples remained relatively stable within the concentration range of 10-16 mg·mL~(-1)( total injection amount of 100-160 μg). The RSDs of the percentage of polymers were 0. 00%( n = 3,10 mg·mL~(-1)),0. 10%( n = 3,12 mg·mL~(-1)),and 0. 10%( n = 3,16 mg·mL~(-1)),respectively. The UPLC method was used to determine the national control sample for human albumin of 12 mg·mL~(-1),and the mean value of peak area%was 5. 62%( n = 10). The results were consistent with those of the parallel determination by HPLC( 5. 58%),both of which were in accordance with the quality control range of the national standard for human albumin. The RSD of the percentage of the peak area of the polymers in national standard for human albumin by UPLC was 0. 40%( n = 10). The HPLC and UPLC methods were used to determine the polymer peak area percentage of 20 batches of HSA samples from 7 domestic and foreign enterprises at the concentration of 12 mg·mL~(-1). The correlation coefficient of the two methods was 0. 996 0( P > 0. 05) and there was no significant difference between the two methods( P > 0. 05). CONCLUSION Compared with the traditional HPLC method,the detection time of HSA SEC by the proposed UPLC method is shortened by at least 10 times,and the accuracy and repeatability of the determination are satisfactory. UPLC method can save much more analysis time,is simple and much faster,and can be used for high-throughput determination of molecularsize distribution of human serum albumin.
引文
[1]China Food and Drug Administration.Announcement of the China Food and Drug Administrative measures for the batch release of biological products(decree No.39 of the State Food and Drug Administration)[EB/OL].[2017-12-29]http://www.sfda.gov.cn/WS01/CL0053/220852.html
[2]Ch.P(2015)VolⅢ(中国药典2015年版.三部)[S].2015:244-246.
[3]WANG M L,XIAO L,ZHOU Q,et al.Preparation of the first Chinese national standard for human albumin[J].Chin Pharm J(中国药学杂志),2017,52(6):480-487.
[4]ZHANG D,CHEN B.Trend and preliminary analysis of the polymer in the human albumin production process[J].Chin J Blood Transfus(中国输血杂志),2016,29(9):909-912.
[5]EP 9.0.Human albumin solution[S].2660-2662.
[6]US.P 39.Albumin Human[S].2352-2353.
[2]Ch.P(2015)VolⅢ(中国药典2015年版.三部)[S].2015:244-246.
[3]WANG M L,XIAO L,ZHOU Q,et al.Preparation of the first Chinese national standard for human albumin[J].Chin Pharm J(中国药学杂志),2017,52(6):480-487.
[4]ZHANG D,CHEN B.Trend and preliminary analysis of the polymer in the human albumin production process[J].Chin J Blood Transfus(中国输血杂志),2016,29(9):909-912.
[5]EP 9.0.Human albumin solution[S].2660-2662.
[6]US.P 39.Albumin Human[S].2352-2353.