Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.

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

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.

Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies.

Within pharmaceutical industry charge heterogeneity testing of biopharmaceuticals has to be reproducible and fast. It should pass method validation according to ICH Q2. Classical approaches for the analysis of the charge heterogeneity of biopharmaceuticals are ion exchange chromatography (IEC) and isoelectric focusing (IEF). As an alternative approach, also capillary zone electrophoresis (CZE) was expected to allow reliable charge heterogeneity profiling by separation according to the analyte's net charge and hydrodynamic radius. Aim of this study was to assess if CZE possesses all of the required features. Therefore, beside lab internal validation of this method also an international cross company study was organized. It was shown that CZE is applicable across a broad pI range between 7.4 and 9.5. The coefficient of correlation was above 0.99 which demonstrated linearity. Precision by repeatability was around 1% (maximum relative standard deviation per level) and accuracy by recovery was around 100% (mean recovery per level). Accuracy was further verified by direct comparison of IEC, IEF and CZE, which in this case showed comparable %CPA results for all three methods. However, best resolution for the investigated MAb was obtained with CZE. In dependence on sample concentration the detection limit was between 1 and 3%. Within the intercompany study for CZE the same stressed and non-stressed samples were analyzed in each of the 11 participating labs. The finally obtained dataset contained more than 1000 separations which provided an extended dataset for further statistical evaluation. Among the different labs no significant differences between the peak profiles were observed. Mean driver for dropouts in quantitative evaluation was linked to the performance of some participating labs while the impact of the method performance was negligible. In comparison to a 50cm capillary there was a slightly better separation of impurities and drug substance related compounds with a 30cm capillary which demonstrates that an increased stability indicating potential can be combined with the increased separation velocity and high throughput capability of a shorter capillary. Separation can be performed in as little as approx. 3min allowing high throughput applications. The intercompany study delivered precise results without explicit training of the participating labs in the method prior to the study (standard deviations in the range of 1%). It was demonstrated that CZE is an alternative platform technology for the charge heterogeneity testing of antibodies in the pharmaceutical industry.