目的 优化2015年版《中国药典》已收录的十二烷基硫酸钠毛细管电泳法(capillary electrophoresis-sodium dodecyl sulfate,CE-SDS)用于单抗分子大小变异体测定,及制备CE-SDS的系统适用性对照品。方法 优化2015年版《中国药典》CE-SDS的关键参数,包括样品缓冲液的pH及紫外检测波长;制备了CE-SDS的系统适用性对照品,开展了多个实验室间协作研究,并利用分子排阻色谱(size exclusion-high performance liquid chromatography, SEC-HPLC)、成像毛细管等电聚焦电泳(imaged capillary isoelectric focusing, iCIEF)及CE-SDS评价其长期稳定性(18个月)。结果 优化了该方法的关键参数:样品缓冲液pH值为6.5,检测波长为214或220 nm;制备了CE-SDS的系统适用性对照品;协作标定非还原CE-SDS的主峰纯度为92.19%～94.77%,还原CE-SDS的非糖基化重链(non-glycosylated heavy chain,NGHC)含量/总重链含量为0.93%～1.66%;18个月的长期稳定性良好。结论 修订了2015年版《中国药典》三部3127:单抗分子大小变异体测定法(CE-SDS法),并为该法研制了系统适用性对照品,收录于2020年版《中国药典》三部3127,助力相关企业的研发和质控。
Abstract
OBJECTIVE To optimize the capillary electrophoresis-sodium dodecyl sulfate(CE-SDS)method of Chinese Pharmacopoeia (ChP) 2015 edition used for size heterogeneity analysis of monoclonal antibody products, and prepare system suitability standard for CE-SDS. METHODS Some key parameters of CE-SDS method in ChP 2015 edition were optimized, such as pH value of sample buffer and wavelengths of UV detection. The system suitability standard for CE-SDS method was prepared and collaborative study for the standard was conducted, while its long-term stability up to 18 m was evaluated by iCIEF, SEC-HPLC and CE-SDS. RESULTS The two key parameters were optimized to be as follows: pH value of sample buffer was 6.5, and wavelengths of UV detection were 214 nm or 220 nm. The system suitability standard for CE-SDS was successfully prepared. In the collaborative study for the standard, the main peak purity in non-reduced CE-SDS was 92.19%-94.77% and the ratios of NGHC to total heavy chain in reduced CE-SDS was 0.93%-1.66%. The long-term stability up to 18 m was confirmed. CONCLUSION In this study, determination of molecular size heterogeneities inmonoclonal antibodies (CE-SDS method) of Chinese Pharmacopoeia 2015 is revised, including a prepared system suitability standard for the revised method, which is adopted by Chinese Pharmacopoeia 2020 and was helpful for development and quality control of drug companies.
关键词
单克隆抗体 /
大小异质性 /
十二烷基硫酸钠-毛细管电泳 /
系统适用性对照品
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Key words
monoclonal antibody /
size heterogeneity /
CE-SDS /
system suitability standard
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中图分类号：
R917
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参考文献
[1] MARSTON H D, PAULES C I, FAUCI A S. Monoclonal antibodies for emerging infectious diseases — borrowing from history[J]. N Engl J Med, 2018, 378(16):1469-1472.
[2] BOYIADZIS M, FOON K A . Approved monoclonal antibodies for cancer therapy[J]. Expert Opin Biol Ther, 2008, 8(8):1151-1158.
[3] HERSCHEL T, EL-ARMOUCHE A, WEBER S. Monoclonal antibodies, overview and outlook of a promising therapeutic option[J]. Dtsch Med Wochenschr, 2016, 141(19):1390-1394.
[4] LOWE D, DUDGEON K, ROUET R, et al. Aggregation, stability, and formulation of human antibody therapeutics[J]. Adv Protein Chem Struct Biol, 2011, 84(84):41-61.
[5] ROBERTS C J. Therapeutic protein aggregation: mechanisms, design, and control[J]. Trends Biotechnol, 2014, 32(7):372-380.
[6] LIU H, GAZA-BULSECO G, FALDU D, et al. Heterogeneity of monoclonal antibodies[J]. J Pharm Sci, 2008, 97(7):2426-2447.
[7] ZHU L, SHAN G X, WANG L. Effect of glycosylation modification on the stability of recombinant CTLA4-Ig[J]. Chin J Pharm Anal(药物分析杂志), 2019,39(1):70-77.
[8] WEI W, LUO J H, YIN H Z, et al. Comments on the development and assessment of the drug impurities and substances related to recombinant monoclonal antibodies[J]. Chin J New Drugs(中国新药杂志), 2014, 23(8):906-911.
[9] VLASAK J, IONESCU R. Fragmentation of monoclonal antibodies[J]. MAbs, 2011, 3(3):253-263.
[10] TUMER A, YANDROFSKI K, TELIKEPALLI S, et al. Development of orthogonal NISTmAb size heterogeneity control methods[J]. Anal Bioanal Chem, 2018, 410(8):2095-2110.
[11] YU C F, WANG W B, LI M, et al. Size heterogeneity analysis of monoclonal antibody products[J]. Chin J Microbiol Immunol(中华微生物学和免疫学杂志), 2014, 34(9):718-722.
[12] SHI Y, LI Z, LIN J. Advantages of CE-SDS over SDS-PAGE in mAb purity analysis[J]. Anal Bioanal Chem, 2012, 4(6):1637-1642.
[13] ZHANG J, BURMAN S, GUNTURI S, et al. Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody[J]. J Pharm Biomed Anal, 2010, 53(5):1236-1243.
[14] LI M, YU C F, WANG W B, et al. Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs[J]. Electrophoresis, 2021, 42(19):1900-1913.
[15] ICH Harmonised Tripartite Guideline Validation of Analytical Procedures:Text and Methodology Q2 (R1) (Current Step 4 Version Dated November 2005) [EB/OL]. 2005: 10-11. https://www.ich.org/products/guidelines/quality/article/quality-guidelines.html.
[16] WU G, YU C F, WANG W B, et al. Methodological validation of imaging capillary electrofocusing electrophoresis for analysis of charge heterogeneity of monoclonal antibodies and preparation of system suitability control materials[J]. Chin J Pharm Anal (药物分析杂志), 2019, 39(1):62-69.
[17] Ch.P (2020) Vol Ⅲ (中国药典2020年版. 三部) [S]. 2020: 502-503.
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脚注
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基金
国家药典标准提高课题项目资助(2018S003)
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