Higel Fabian, Seidl Andreas, Demelbauer Uwe, Viertlboeck-Schudy Margot, Koppenburg Vera, Kronthaler Ulrich, Sörgel Fritz, Friess Wolfgang
Analytical Characterization, Sandoz Biopharmaceuticals, HEXAL AG, Keltenring 1+3, 82041, Oberhaching, Germany.
Process Analytics, Sandoz Biopharmaceuticals, Schaftenau, Austria.
Pharm Res. 2015 Nov;32(11):3649-59. doi: 10.1007/s11095-015-1724-0. Epub 2015 May 28.
In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.
Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program.
The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate.
The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.
本研究提出了一种创新的、高灵敏度的工作流程，该流程能够在临床前开发阶段就分析个体糖变体对药代动力学可能产生的影响。糖变体对药代动力学的可能影响已经成为多项研究的主题，部分结果相互矛盾，这可能与研究设置的差异有关。
使用基于96孔板的亲和纯化方法从临床前样品中分离出一种IgG1抗体，并通过纳升液相色谱-质谱联用仪对聚糖进行单独分析。前提是要有基于稳定重同位素标记聚糖的参考标准品。高灵敏度和低样品消耗使得该方法能够整合到临床前开发计划中。
一项临床前兔研究中一种IgG1生物药物的数据表明，与通过酶联免疫吸附测定法确定的所有分子变体的平均值相比，一些N-糖型具有不同的药代动力学特征。IgG1高甘露糖糖型M5和M6从循环中清除的速率更高。
临床前研究结果证明了所开发的创新工作流程的适用性。可以单独确定糖变体的药代动力学特征。得出的结论是，M6在循环中被甘露糖苷酶转化为M5，而M5又通过与甘露糖受体结合被选择性清除，这与先前发表的结果一致。因此，所开发的技术能够提供可靠的结果，可应用于其他单克隆抗体和其他类型生物药物的药代动力学分析。
