重Analytical EquipmentHIGH PERFORMANCECAPILLARY ELECTROPHORESISSYSTEMwww.lumexinstruments.ccm RECOMBINANT THERAPEUTIC MONOCLONAL ANTIBODIES. DETERMINATION OFPRODUCT-RELATED IMPURITIES ACCORDING TO USP 129 AND CH.P 3127 INTRODUCTION The present method is used for the determination of product-related impurities in recombinant therapeuticmonoclonal antibodies (Immunoglobulin G, IgG) according to United States Pharmacopoeia generalchapter 129 (Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies, Capillary SDSelectrophoresis) using CAPEL-205 capillary electrophoresis system. Alternatively, the determination of lgGimpurities in accordance with Chinese Pharmacopoeia general chapter 3127 (Monoclonal antibodies -Determination of molecular size variants (CE-SDS)) can also be performed using CAPEL-205. MEASUREMENT METHOD The measurement method is based on capillary gel electrophoresis (CGE) with direct UV detection at thewavelength of 220 (214) nm. Sample preparation procedure (denaturation with SDS under reducing or non-reducing conditions) and analysis conditions in accordance with USP 129 or Ch.P 3127. EQUIPMENT AND REAGENTS The CAPEL-205 capillary electrophoresis system is used in measurements. Data acquisition, collection,processing and output are performed using a personal computer running under WINDOWS@ XP/7/8/10operating system with installed dedicated software package ELFORUN. All reagents according to USP 129 or Ch.P 3127. EXAMPLES OF REAL ANALYSESUSP 129. Reducing conditions -24 mAUHCBGE: gel according to USP 129Capillary: Ltot =30 cm, ID=50 pm LCInjection: -5 kV x 20 secVoltage: -15kV NGTemperature: 25°CISDetection: 220 nm19 20 minSample: Recombinant lgG preparationIS - internal standard:NGLc-light chainNG-non-glycosylated heavy chainHC -heavy chain0 5 10 15 20 25 30 35 minUSP 129. Nonreducing conditions 40 mAU IgGBGE: gel according to USP 129Capillary: -tot=30 cm, ID= 50 pmInjection: -5kVx20 secVoltage: -15kVTemperature: 25°CDetection: 220 nm ISSample: Recombinant lgG preparation F6F4 F22F1IS -internal standard F8 F5 F1-F8-IgG-related impurities (fragments)IgG - intact antibody 0 5 10 15 20 25 30 35 min The contents on this paper are subject to change without notice.To get more specific information, please contact the representative by sales@lumexinstruments.com 背景介绍 重组生物技术制备的单克隆抗体(以下简称重组单抗)是生物制品中一类重要的药物类别。近年来,由于其精确的靶向杀伤和中和等生物学效应,重组单抗在肿瘤、自身免疫性疾病等方面受到了广泛的研究和应用,并且在神经学、眼科学等其他疾病领域的研究也开始逐步发展起来。目前,国内生产的重组单抗主要采用真核细胞培养表达的方法,在工艺研发和质量研究方面,药品企业对主要有效成分的结构研究和质量控制关注较多,但是对产品组成中的杂质和相关物质研究较少或关注度不够。考虑到这些杂质或相关物质是重组单抗产品中的重要组成部分,将会影响到产品的质量、临床应用的安全性和有效性。 本方法主要采用CAPEL-205高效毛细管电泳仪测定重组治疗性单克隆抗体(免疫球蛋白G,IgG)中产物的相关杂质,参照的是美国药典第129章(Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies, Capillary SDS electrophoresis) 中的方法,也可以参照中国药典2015版三部三部通则3127( 单抗分子大小变异体测定法)中的方法。1测试条件USP129.还原条件BGE:按照USP 129 中的凝胶条件毛细管电泳: Ltot = 30 cm, ID= 50 µm样品进样: -5 kV x 20 sec电压: -15 kV室温: 25 ОС检测波长: 220 nm 样品: 重组IgG制剂 IS – 内标 LC – 轻链 NG – 非糖基化重链 HC – 重链USP129.非还原条件BGE:按照USP 129 中的凝胶条件毛细管电泳: Ltot = 30 cm, ID= 50 µm样品进样: -5 kV x 20 sec电压: -15 kV室温: 25 ОС检测波长: 220 nm 样品:重组IgG制剂 IS - 内标 F1-F8 - IgG相关杂质(片段) IgG - 完整抗体2测试结果图1 还原条件下的检测色谱图图2 非还原条件下的 检测色谱图3结论 采用CAPEL-205高效毛细管电泳仪测定重组治疗性单克隆抗体(免疫球蛋白G,IgG)中产物的相关杂质,可以实现的IgG产物中的相关杂质的有效分离,方法准确、灵敏度高、重现性好,可以满足日常分析要求。4配置列表仪器配置CAPEL-205毛细管电泳系统,装有WINDOWS®XP/ 7/8/10操作系统的笔记本电脑ELFORUN专用软件工作站Capel-205毛细管电泳仪的优势特点通过施加高压电场,基于毛细管液体样本组成成分迁移速度的差别。应用UV吸收检测技术,在毛细管出口应用高精度光学系统定量检测分离出的组分 光源:高强度氘灯,优化光学设计保证低检出限 可实现多种不同分析方法和分离模式,毛细管区带电泳,毛细管胶束电动色谱,毛细管等电聚集,毛细管凝胶电泳,毛细管等速电泳 全自动进样,进样方式可选电动及压力进样。压力进样,步进值1mbar,最高99mbar;电动进样,电压范围:步进值1kV,1-25kV可调毛细管冷却:恒温水冷系统,环境温度5-50℃可调;恒温水冷保证温度控制更加精细和准确,提高分析效果(相对与风冷,冷却效果更优/与液冷相比,成本更低) 标准离心管1.5mL(Eppendorf型)适用于所有样本分析 扩展的全自动进样器,高达59个进样位,允许提供自动过夜分析功能 独特全自动开口进样设计,避免挥发和交叉污染 新型高压模块提供±30kV压力,保证超优的分析效率和快速分析时间 采用流动电位控制技术,极大提高重现性,尤其是分析复杂样品更具优势