Microchip assays for screening monoclonal antibody product quality

被引:42
作者
Chen, Xiaoyu [1 ]
Tang, Kaiyan [1 ]
Lee, Maximilian [1 ]
Flynn, Gregory C. [1 ]
机构
[1] Amgen Inc, Analyt & Formulat Sci, Thousand Oaks, CA 91320 USA
关键词
CE-SDS; High-throughput analysis; Microchip; Monoclonal antibody; Quality screening;
D O I
10.1002/elps.200800324
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Microchip CE-SDS was evaluated as a high-throughput alternative to conventional CE-SDS for monitoring monoclonal antibody protein quality. A commercial instrument (LabChip (R) 90) was used to separate dodecyl sulfate coated proteins through a sieving polymer based on the proteins' sizes. Under reducing conditions, the microchip CE-SDS separation was similar to that of conventional CE-SDS, providing reasonable resolution of the non-glycosylated and the glycosylated heavy chains. The fluorescence detection on LabChip 90 using non-covalent fluorescent labeling method was about as sensitive as the 220 nm UV detection used in a conventional CE instrument. A simple glycan typing assay was developed for the reducing microchip CE-SDS format. Antibodies, either pure or in crude cell culture media are treated with Endoglycosidase H, which specifically cleaves the hybrid and high mannose type glycans. A heavy chain migration shift on reducing CE-SDS resulting from the loss of glycan is used to measure the level of high mannose/hybrid type glycans as a percentage of the total glycans. Microchip CE-SDS, under both non-reducing and reducing conditions, can be used in a variety of antibody product screening assays. The microchip analyses provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation under typical operational conditions.
引用
收藏
页码:4993 / 5002
页数:10
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