Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs.

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation.

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell-mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

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.