A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current... Show moreA broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Show less
High throughput methods for oligosaccharide analysis are required when searching for glycan based biomarkers Next to mass spectrometry based methods which allow fast and reproducible analysis of... Show moreHigh throughput methods for oligosaccharide analysis are required when searching for glycan based biomarkers Next to mass spectrometry based methods which allow fast and reproducible analysis of such compounds further separation based techniques are needed which allow for quantitative analysis Here an optimized sample preparation method for N glycan profiling by multiplexed capillary gel electrophoresis with laser induced fluorescence detection (CGE LIF) was developed, enabling high throughput glycosylation analysis First, glycans are released enzymatically from denatured plasma glycoproteins Second, glycans are labeled with APTS using 2-picoline borane as a nontoxic and efficient reducing agent Reaction conditions are optimized for a high labeling efficiency short handling times and only limited loss of sialic acids Third samples are subjected to hydrophilic interaction chromatography (HILIC) purification at the 96 well plate format Subsequently, purified APTS labeled N glycans are analyzed by CGE LIF using a 48-capillary DNA sequencer The method was found to be robust and suitable for high-throughput glycan analysis Even though the method comprises two overnight incubations, 96 samples can be analyzed with an overall labor allocation time of 2 5 h The method was applied to serum samples from a pregnant woman, which were sampled during first second and third trimesters of pregnancy, as well as 6 weeks 3 months, and 6 months postpartum Alterations in the glycosylation patterns were observed with gestation and time after delivery Show less
