Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

Abstract

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

Fc Sialylation Prolongs Serum Half-Life of Therapeutic Antibodies

“…By contrast, this SNA reactivity was absent for the WT IgG as well as the C6A-74 and T5A-74 variants, which do not include the Del mutation. The composition of the Fc N-glycans was assessed using two complementary approaches: first, high-performance CE laser-induced fluorescence (HPCE-LIF) (48,49) and second, an orthogonal technique based on liquid chromatography (LC) and mass spectrometry (MS) (44). For HPCE-LIF analyses, N-glycans were released by PNGase F treatment (Fig.…”

Multi‐site N‐Glycan mapping study 2: UHPLC

“…N‐glycosylation is recognized as being one of the key product quality indicators by regulatory agencies and therefore requires such methods. The appropriateness and capability of capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) were previously established and reported . Parallel UHPLC‐HILIC results from the same international study are presented here.…”

“…All peaks with ≥0.1% peak area were evaluated and outlier analysis was performed using Grubb's and Cochran's C tests. As performed previously for the CE‐LIF study , standard statistical analysis (repeatability and reproducibility) as well as outlier analysis were performed for all participating sites based on ISO 5725‐2 guidelines . Repeatability (intra‐laboratory precision) and reproducibility (inter‐laboratory precision) were assessed for relative peak areas for each site.…”

“…Repeatability (intra‐laboratory precision) and reproducibility (inter‐laboratory precision) were assessed for relative peak areas for each site. Critical examination for outlier identification was performed as previously discussed by: (i) laboratories deviating in precision (repeatability) indicating laboratory bias; (ii) individual results at laboratories deviating in either precision or in mean values.…”

Recent Advances in the Analysis of Complex Glycoproteins

“…Recently, 20 independent laboratories representing biopharmaceutical companies, contract laboratories, academic institutions, and regulatory agencies participated in a comparative analytical trial on the N-glycan analysis by CE-LIF with highly satisfactory results; a close comparison with a competitive method (UHPLC) is reportedly in progress, but the results were not available at this time as of yet. 189 Additional aspects of biopharmaceutically important applications of CE-LIF profiling and quantification, such as sample preparation, were also addressed. 190 While these methodologies usually concern the glycan sample components with known or predictable structure, there is still a need for authentic glycan standards and N-glycan databases in this area.…”