基于硼酸亲和色谱(BAC)测定单抗糖化比例方法的建立

刘旭梅; 王文波; 武刚; 于传飞; 韩治国; 王兰; 刘万卉

文章摘要

刘旭梅,王文波,武刚,于传飞,韩治国,王兰,刘万卉.基于硼酸亲和色谱(BAC)测定单抗糖化比例方法的建立[J].中国药事,2023,(3):287-297

基于硼酸亲和色谱(BAC)测定单抗糖化比例方法的建立

Establishment of Quantitative Method for Determination of Monoclonal Antibody Glycation Level Based on Boronate Affi nity Chromatography (BAC)

DOI：10.16153/j.1002-7777.2023.03.006

中文关键词: 单克隆抗体利妥昔单抗糖化硼酸亲和色谱方法学验证

英文关键词: monoclonal antibody rituximab glycation boronate affinity chromatography methodology validation

基金项目:《中国药典》药品标准提高课题项目资助(编号 2020S08)

作者	单位
刘旭梅	烟台大学药学院,烟台 246005 ；中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京 102629
王文波	中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京 102629
武刚	中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京 102629
于传飞	中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京 102629
韩治国	安捷伦科技(北京)有限公司,北京 100102
王兰	中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京 102629
刘万卉	烟台大学药学院,烟台 246005

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中文摘要:

目的：建立基于硼酸亲和色谱(BAC)测定单抗糖化比例分析方法并进行方法学验证。方法：对硼酸亲和色谱做方法优化和方法学验证，通过对流动相的盐组成、NaCl和Tris浓度的优化，建立能够有效定量单抗糖化比例的BAC分析方法，并对该方法进行方法学验证和应用评价。结果：NaCl和Tris浓度的变化会影响单抗糖化比例的检测，通过参数优化，最终确定在NaCl和Tris浓度分别为300 mmol·L^-1 和25 mmol·L^-1时，可以对单抗糖化比例进行准确定量。根据ICH Q2和《中华人民共和国药典》2020 年版对建立的方法进行了方法学验证，结果表明该方法具有良好的特异性、准确性、精密度和耐用性。单抗糖化比例在2%~90%有良好的线性，R²大于0.99，回收率在87.04%~115.29%；非糖化比例在 10%~98%具有良好线性，R² 大于0.99，回收率在99.69%~109.79%。精密度结果表明，该方法整体的 RSD值小于18.26%。最后，利用建立的方法对利妥昔单抗原研药和生物类似药进行了糖化比例检测，发现同一厂家不同批次利妥昔单抗糖化比例差异较小，而不同厂家糖化比例存在差异。结论：建立的BAC 糖化比例测定方法具有良好的准确性和精密度，可用于利妥昔单抗糖化比例的检测。

英文摘要:

Objective: To establish and validate an analytical method based on boronate affi nity chromatography (BAC) for the determination of glycation level of monoclonal antibody. Methods: The mobile phase composition, including NaCl and Tris concentrations were optimized, and the established BAC method was systematically validated and used for the evaluation of glycation level of rituximab. Results: The change of the concentration of NaCl and Tris affected the determination of glycation level of monoclonal antibody. Through parameter optimization, it was fi nally determined that when the NaCl and Tris concentrations in mobile phase were set to 300 mmol·L^-1 and 25 mmol·L^-1 respectively, the glycation level could be quantifi ed accurately. The established BAC method was validated according to ICH Q2 guideline and 2020 editions of the Chinese Pharmacopoeia. This method demonstrated good specifi city, accuracy, precision, and robustness. Glycation level between 2% and 90% showed good linearity with R²＞0.99, and the recovery rate was between 87.04% and 115.29%. For non-glycation level between 10% and 98% showed good linearity with R² ＞0.99, and the recovery rate was between 99.69% and 109.79%. The precision results showed that the overall RSD value of the method was＜18.26%. Finally, we also evaluated the glycation level of rituximab original drugs and its biosimilars by using the established BAC method, and found that the level of glycation was comparable among diff erent batches from the same manufacturer, but varied in diff erent manufacturers. Conclusion: The established BAC method showed good accuracy and precision, and can be used for glycation level quantitation of rituximab.

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