成像毛细管等电聚焦电泳分析单克隆抗体电荷异质性的方法学验证及系统适用性对照品的制备

目的：采用成像毛细管等电聚焦电泳（imaging capillary electrofocusing electrophoresis，iCIEF）分析单抗的电荷异质性，组织国内多家质控实验室对该方法进行验证；制备用于iCIEF法的系统适用性对照品。方法：邀请19个质控实验室，应用3种品牌（4种型号）的iCIEF设备进行方法学的预验证，确定该方法的关键试剂、仪器参数设置、数据分析要点等；依据ICH_Q2_R1指导原则、2015年版《中华人民共和国药典》（简称《中国药典》）通则9101制定方法学验证方案，邀请11个质控实验室参加验证；制备基于iCIEF法的系统适用性对照品并利用SEC-HPLC、IEC-HPLC、非还原CE-SDS进行稳定性评价。结果：预验证结果显示：等电点标记物（pI Marker）为关键试剂；不同品牌、型号的iCIEF检测设备参数设置无需统一；数据处理可采用自动积分、手动积分相结合的形式。验证结果：完成了该方法的特异性、准确度、精密度、线性及其范围、耐用性、定量下限的评价；制备了iCIEF法的系统适用性对照品，并进行了加速及长期稳定性评价，制定了相应的合格标准。结论：首次组织了iCIEF的方法学验证，为《中国药典》标准提高提供方法学验证依据，为该方法制备了系统适用性对照品。

Objective:To use imaging capillary electrofocusing electrophoresis(iCIEF) for charge heterogeneity analysis of monoclonal antibody products,to organize multiple quality control laboratories in China to carry out validation of the method and prepare system suitability control materials for iCIEF. Methods:Three brands (four models) of iCIEF equipments were used to carry out the pre-validation of methodology of iCIEF,and to determine the key reagents,instrument parameters,data analysis key points of the method in 19 quality control laboratories. According to ICH_Q2_R1 guideline and general principles 9101 of the 2015 edition of the Pharmacopoeia of the People's Republic of China (China Pharmacopoeia),a protocol of validation for methodologies was formulated. Eleven quality control laboratories were invited to participate in the validation;the system suitability control materials based on iCIEF method were prepared and their stability was evaluated by SEC-HPLC,IEC-HPLC and non-reduced CE-SDS. Results:The pre-validation results showed that the isoelectric point marker (pI Marker) was the key reagent,it was not necessary to unify the parameters of iCIEF detection equipment of different brands and models,and data processing could adopt the combination of automatic and manual integration. The methodological validation results:the specificity,accuracy,precision,linearity and range,durability and quantitative limits of the method were evaluated,the system suitability control materials for iCIEF method were successfully prepared,its acceleration and long-term stability were evaluated,and the corresponding qualification standards were formuated. Conclusion:The validation of iCIEF methodology was organized for the first time,to provide a methodological validation basis for the improvement of China Pharmacopoeia standards,and a system suitability control material was prepared for the method.

[1] KOHLER G,MILSTEIN C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].Nature,1975,256(5517):495
[2] CHRISTIANSEN J,RAJASEKARAN AK.Biological impediments to monoclonal antibody-based cancer immunotherapy[J].Mol Cancer Ther,2004,3(11):1493
[3] BREEN ED,CURLEY JG,OVERCASHIER DE,et al.Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation[J].Pharm Res,2001,18(9):1345
[4] GESKIN LJ.Monoclonal antibodies[J].Dermatol Clin,2015,33(4):777
[5] KIRLEY TL,NORMAN AB.Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its fab fragment[J].Hum Vaccin Immunother,2015,11(2):458
[6] DESOUBEAUX G,REICHERT JM,SLEEMAN M,et al.Therapeutic monoclonal antibodies for respiratory diseases:current challenges and perspectives[J].MAbs,2016,8(6):999
[7] ECKER DM,JONES SD,LEVINE HL.The therapeutic monoclonal antibody market[J].MAbs,2015,7(1):9
[8] 于传飞,郭玮,王兰,等.成像毛细管等电聚焦电泳法对单克隆抗体制品的电荷异质性分析[J].药物分析杂志,2014,34(7):1212 YU CF,GUO W,WANG L,et al.Charge heterogeneity analysis of monoclonal antibody products with imaging capillary isoelectric focusing electrophoresis[J].Chin J Pharm Anal,2014,34(7):1212
[9] 王丽,周勇,王军志.成像毛细管等电聚焦电泳分析糖蛋白类生物技术药物[J].中国生物制品学杂志,2012,25(8):1035 WANG L,ZHOU Y,WANG JZ.Analysis of glycoproteins by imaging capillary isoelectric focusing electrophoresis[J]. Chin J Biol,2012,25(8):1035
[10] SOSIC Z,HOUDE D,BLUM A,et al.Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity[J].Electrophoresis,2008,29(21):4368
[11] ANDERSON CL,WANG Y,RUSTANDI RR.Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products[J].Electrophoresis,2012,33(11):1538
[12] SALAS SO,KENNEL B,PARK SS,et al.Robustness of iCIEF methodology for the analysis of monoclonal antibodies:an inter laboratory study[J].J Sep Sci,2012,35(22):3124
[13] WU J,PAWLISZYN J.Application of capillary isoelectric focusing with absorption imaging detection to the quantitative determination of human hemoglobin variants[J].Electrophoresis,1995,16(4):670
[14] DADA OO,JAYA N,VALLIERE DJ,et al.Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation[J].Electrophoresis,2015,36(21-22):2695
[15] WU XZ,PAWLISZYN J.Whole-column imaging capillary electrophoresis of proteins with a short capillary[J].Electrophoresis,2002,23(4):542
[16] SOSIC Z,HOUDE D,BLUM A,et al.Application of imaging capillary IEF for characterization and quantitative analysis of recombinant protein charge heterogeneity[J].Electrophoresis,2008,29(21):4368
[17] RUSTANDI RR,ANDERSON C,HAMM M.Application of capillary electrophoresis in glycoprotein analysis[J].Methods Mol Biol,2013,988:181
[18] JANINI G,SAPTHARISHI N,WASELUS M,et al.Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system[J].Electrophoresis,2002,23(11):1605
[19] WU J,WU XZ,HUANG T,et al.Analysis of proteins by CE,CIEF,and microfluidic devices with whole-column-imaging detection[J].Methods Mol Biol,2004,276:229
[20] Harmonised Tripartite Guideline.Validation of Analytical Procedures:Text and Methodology Q2(R1)(Current Step 4 Version Dated 12 September 2005)[S/OL].[2016-07-01].https://www.ich.org/products/guidelines/quality/article/quality-guidelines.html