目的: 对毛细管电泳结合激光诱导荧光(CE-LIF)检测单抗N糖谱方法进行多家实验室的联合验证。方法:依据国际人用药品注册技术协调会(ICH)Q2_R1指导原则和2015年版《中华人民共和国药典》通则9101,开展方法学验证。评价指标包括专属性、线性、准确度、精密度、定量限、范围和耐用性。结果:验证数据表明,该方法具有良好的特异性、准确性、精密性和耐用性。蛋白量在100~400 μg范围内具有良好的线性,R2大于0.97,且回收率在81%~109%范围内。精密性评价结果显示,G0F、Man5、G1F(1,6)、G1F(1,3)和G2F的校正峰面积百分比和迁移时间的RSD值均小于10%。该方法的定量下限为0.016%,可对在0.016%~77.233%范围内的单个色谱峰进行准确定量分析。同时多个条件下的耐用性评价结果表明该方法具有耐用性,峰面积百分比的RSD小于9%,迁移时间的RSD小于0.1%。结论:组织开展了基于CE-LIF的检测单抗N糖谱的方法学联合验证,结果表明方法可行,可用于单抗N糖谱的检测。
Objective: To validate the CE-LIF method for N-glycan profile analysis of antibodies inter laboratory. Methods: The method validation was performed according to ICH_Q2_R1 guideline and general chapter 9101 of Chinese Pharmacopoeia (2015 edition). The validation characteristics include specificity, linearity, accuracy, precision, quantitation limit, range and robustness. Results: The method showed good specificity, accuracy, precision and robustness, and showed a good linearity at a protein content ranging from 100 μg to 400 μg. The R2 of linear regression equation were above 0.97, and the recoveries were between 81% and 109%. For precision test, the RSDs of peak area percentage and retention time of G0F, Man5, G1F(1,6), G1F(1,3) and G2F were below 10%. The quantitation limit of the method was 0.016%, and the range was from 0.016% to 77.233%, whichindicated that single peak at this range could be quantified accurately. Furthermore, robustness evaluation under a series of condition showed that this method was robust, where the RSD of peak area percentage was below 9% and RSD of retention time was below 0.1%. Conclusion: Inter-laboratory method validation of CE-LIF has been organized, and the results verifies that the method is feasible for analysis of antibodies.
[1] CHUNG AW, CRISPIN M, PRITCHARD L, et al. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function[J]. AIDS, 2014, 28(17):2523
[2] JEFFERIS R. Glyco-engineering of human IgG-Fc to modulate biologic activities[J].Curr Pharm Biotechnol, 2016, 17(15):1333
[3] LIU L. Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins[J].J Pharm Sci, 2015, 104(6):1866
[4] PACE D, LEWIS N, WU T, et al. Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcgammaRIIIa receptor binding activity of an IgG1 antibody[J].Biotechnol Prog, 2016, 32(5):1181
[5] ZHENG K, YARMARKOVICH M, BANTOG C, et al. Influence of glycosylation pattern on the molecular properties of monoclonal antibodies[J].MAbs, 2014, 6(3):649
[6] SHEN Y, SMITH RD. Proteomics based on high-efficiency capillary separations[J].Electrophoresis, 2002, 23(18):3106
[7] MANABE T. Capillary electrophoresis of proteins for proteomic studies[J].Electrophoresis, 1999, 20(15-16):3116
[8] GUTTMAN A. Capillary sodium dodecyl sulfate-gel electrophoresis of proteins[J].Electrophoresis, 1996, 17(8):1333
[9] GUTTMAN A, NOLAN J. Comparison of the separation of proteins by sodium dodecyl sulfate-slab gel electrophoresis and capillary sodium dodecyl sulfate-gel electrophoresis[J].Anal Biochem, 1994, 221(2):285
[10] BWANALI L, CRIHFIELD CL, NEWTON EO, et al. Quantification of the alpha 2-6 sialic acid linkage in branched N-glycan structures with capillary nanogel electrophoresis[J].Anal Chem, 2020, 92(1):1518
[11] LU G, HOLLAND LA. Profiling the N-glycan composition of igg with lectins and capillary nanogel electrophoresis[J].Anal Chem, 2019, 91(2):1375
[12] MESZAROS B, KOVACS Z, GEBRI E, et al. N-glycomic analysis of z(iga1) partitioned serum and salivary immunoglobulin a by capillary electrophoresis[J].Curr Mol Med, 2020, 20(10):781
[13] SZEKRENYES A, PARK SS, SANTOS M, et al. Multi-site N-glycan mapping study 1:capillary electrophoresis-laser induced fluorescence[J].MAbs, 2016, 8(1):56
[14] 王文波, 武刚, 于传飞, 等. 高效阴离子色谱结合脉冲安培检测器分析单抗N糖谱的方法学联合验证[J].中国新药杂志, 2020, 29(14):1654
WANG WB, WU G, YU CF, et al. Interlaboratory validation of HPAEC-PAD method for N-glycan profile analysis of monoclonal antibodies[J].Chin J New Drugs, 2020, 29(14):1654
[15] 王文波, 武刚, 于传飞, 等. 亲水相互作用超高效液相色谱法分析单抗N糖谱的方法学联合验证[J].中国药学杂志, 2019, 54(24):2028
WANG WB, WU G, YU CF, et al. Interlaboratory validation of HILIC-UPLC method for N-glycan profile analysis of monoclonal antibodies[J].Chin Pharm J, 2019, 54(24):2028
[16] DAVID NP, NICHOLAS T, SAI RV, et al. Multivariate data analysis of growth medium trends affecting antibody glycosylation[J].Biotechnol Prog, 2020, 36(1):e2903
[17] NITIN A, ALISON M, RAHUL P, et al. Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics[J].Biotechnol Prog, 2019, 35(5):e2865
