Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

Abstract

CZE is a well‐established technique for charge heterogeneity testing of biopharmaceuticals. It is based on the differences between the ratios of net charge and hydrodynamic radius. In an extensive intercompany study, it was recently shown that CZE is very robust and can be easily implemented in labs that did not perform it before. However, individual characteristics of some examined proteins resulted in suboptimal resolution. Therefore, enhanced method development principles were applied here to investigate possibilities for further method optimization. For this purpose, a high number of different method parameters was evaluated with the aim to improve CZE separation. For the relevant parameters, design of experiments (DoE) models were generated and optimized in several ways for different sets of responses like resolution, peak width and number of peaks. In spite of product specific DoE optimization it was found that the resulting combination of optimized parameters did result in significant improvement of separation for 13 out of 16 different antibodies and other molecule formats. These results clearly demonstrate generic applicability of the optimized CZE method. Adaptation to individual molecular properties may sometimes still be required in order to achieve optimal separation but the set screws discussed in this study [mainly pH, identity of the polymer additive (HPC versus HPMC) and the concentrations of additives like acetonitrile, butanolamine and TETA] are expected to significantly reduce the effort for specific optimization.

Application of affinity capillary electrophoresis for charge heterogeneity profiling of biopharmaceuticals

“…It has been shown that the BGE used here is close to the optimum for charge heterogeneity profiling of mAbs . However, peak deteriorations during initial experiments conducted with a mixture of antibody and antigen (data not shown) led to the assumption that there is a strong capillary wall adhesion of the antigen under separation conditions as results were similar to other publications on this problem .…”

“…These compounds prevent excessive Joule heating, reduce EOF and dynamically coat the inner capillary wall. Further investigations with different mAbs have demonstrated the generic applicability of this method and have shown some important parameters for the development of tailor‐made adaptions . An international cross‐laboratory study has demonstrated that CZE is a simple, fast, reliable, and reproducible method for the relative quantification of different charge species .…”

Design of experiments as a valuable tool for biopharmaceutical analysis with (imaged) capillary isoelectric focusing

“…Design of experiments (DoE) is usually part of such a concept and serves as a valuable tool to gain maximal knowledge about adjustable factors within a defined range. In addition, DoE is increasingly used for other analytical tasks, especially for method optimization .…”

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

“…In parallel, increasing efforts have been made to directly hyphenate IEC , CZE and recently iCIEF to mass spectrometry (MS), to quickly identify charge variants at the mAb intact level. However, compromises on the separation performance are often made when directly coupling the LC and CE methods to MS, which explain why CZE‐UV methods are still being developed and applied in the biopharmaceutical industry .…”

“…The EOF and protein adsorption can be limited with (i) the use of static capillary wall coatings or more generally with (ii) a dynamic coating . Most of the CZE methods listed in the review of Gahoual et al .…”