High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated LC-MS-Based Solution

Abstract

Protein glycosylation can impact the efficacy and safety of biotherapeutics and therefore needs to be well characterized and monitored throughout the drug product life cycle. Glycosylation is commonly assessed by fluorescent labeling of released glycans, which provides comprehensive information of the glycoprofile but can be resource-intensive regarding sample preparation, data acquisition, and data analysis. In this work, we evaluate a comprehensive solution from sample preparation to data reporting using a liquid chromatography–mass spectrometry (LC-MS)-based analytical platform for increased productivity in released glycan analysis. To minimize user intervention and improve assay robustness, a robotic liquid handling platform was used to automate the release and labeling of N-glycans within 2 h. To further increase the throughput, a 5 min method was developed on a liquid chromatography–fluorescence–mass spectrometry (LC-FLR-MS) system using an integrated glycan library based on retention time and accurate mass. The optimized method was then applied to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. Consistent relative abundance of critical species such as high mannose and sialylated glycans was obtained for samples within the same batch (mean percent relative standard deviation [RSD] = 5.3%) with data being acquired, processed, and reported in an automated manner. The data acquisition and analysis of the 48 samples were completed within 6 h, which represents a 90% improvement in throughput compared with conventional LC-FLR-based methods. Together, this workflow facilitates the rapid screening of glycans, which can be deployed at various stages of drug development such as process optimization, bioreactor monitoring, and clone selections, where high-throughput and improved productivity are particularly desired.

Process analytics 4.0: A paradigm shift in rapid analytics for biologics development

“…[47][48][49] With the improvement of the mass spectrometry-based detection method and the automation of data analysis, Zhang and coworkers reported about 90% improvement in throughput compared to conventional manual methods. 47 CE-SDS is another common analytical technique for process sample testing which involves multiple sample preparation techniques such as denaturation, reduction, alkylation, and clean up with significant manual labor. 50 Le et al, reported for the first time an automated CE-SDS sample preparation platform which essentially eliminates all the manual steps such as concentration normalization, removal of salts and other contaminants, and addition of the required CE-SDS reagents.…”

“…46 Due to the complexity and laborintensive nature of manual glycan analysis methods a variety of sample preparation automation techniques has been reported recently. [47][48][49] With the improvement of the mass spectrometry-based detection method and the automation of data analysis, Zhang and coworkers reported about 90% improvement in throughput compared to conventional manual methods. 47 CE-SDS is another common analytical technique for process sample testing which involves multiple sample preparation techniques such as denaturation, reduction, alkylation, and clean up with significant manual labor.…”

“…Yang and coworkers demonstrated an automated sample preparation workflow for N‐Glycan analysis by utilizing specialized pipet tips to immobilize the protein of interest followed by enzymatic derivatization of captured glycoproteins and elution of N‐glycans 46 . Due to the complexity and labor‐intensive nature of manual glycan analysis methods a variety of sample preparation automation techniques has been reported recently 47–49 . With the improvement of the mass spectrometry‐based detection method and the automation of data analysis, Zhang and coworkers reported about 90% improvement in throughput compared to conventional manual methods 47 .…”

Carbohydrate Structure Analysis: Methods and Applications

“…Shajahan et al 5 developed a high-throughput method for permethylation of released glycans and propose an automated MSn workflow for the rapid structure characterization of glycoproteins. Zhang et al 6 likewise put forth an efficient high-throughput method for glycomics analysis featuring automated sample preparation, rapid liquid chromatography with fluorescence and mass spectrometric detection, and MS-based target screening for quality control of therapeutic antibody N -glycans. Zhang et al 7 developed O -benzylhydroxylamine (BHA) as a cleavable tag that is both hydrophobic for chromatographic separation of glycans and chromogenic for detection of glycans, in order to obtain sufficient amounts of glycans for functional studies using glycan arrays.…”

USP Reference Standard Monoclonal Antibodies: Tools to Verify Glycan Structure

“…Glycan characterization is commonly performed by analysis of glycans released from the protein backbone. As a result of lacking UV absorption chromophores, the release glycans are usually derivatized by introducing fluorescence tags such as 2-aminobenzamide (2-AB) and 2-aminoanthranilic acid (2-AA) to facilitate High-Performance Liquid Chromatography—Fluorescent Detection (HPLC-FLR) [ 10 ]. HPLC-FLR is suitable for profiling and quantitative analysis but is limited for structure identification [ 10 , 11 ].…”