Multiplexing N‐glycan analysis by DNA analyzer

Analysis of N‐glycan structures has been gaining attentions over the years due to their critical importance to biopharma‐based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N‐glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio‐safety and bio‐activity. The ability to comprehensively characterize highly complex mixtures of N‐glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time‐consuming sample pretreatment procedures. CE‐LIF is one of the typical techniques for N‐glycan analysis due to its high separation efficiency. In this paper, a 16‐capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N‐glycan assay throughput. Routinely, the labeling dye used for CE‐LIF is 8‐aminopyrene‐1,3,6‐trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross‐validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross‐validation. The optimized method combines the parallel separation capacity of multiple‐capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N‐glycans. These new methods provided enough useful structural information to permit N‐glycan structure elucidation with only one sample injection.