Dual‐detection approach for a charge variant analysis of monoclonal antibody combination products using imaged capillary isoelectric focusing

Abstract

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…CIEF and iCIEF have matured into techniques that are routinely applied in analytical laboratories, especially in the biopharmaceutical industry. Exemplary applications include, but are not limited to, monoclonal antibodies [192,[209][210][211][212][213][214][215], bispecific antibodies [216][217][218], ADCs [219,220], PEGylated proteins [221,222], glycoproteins, in particular erythropoietin [213,223,224], and fusion proteins [213,225]. Because this subsection is part of the former "proteins" section, which was extended to all biopharmaceuticals, it should not go unmentioned that the range of applications has been expanded beyond proteins and includes oligosaccharides [226], LNPs and related mRNA vaccines [227][228][229][230], virus-like particles [231,232], poliovirus [230,233], adeno-associated virus [230,[234][235][236][237], papillomavirus [238], and bacteria and yeast cells [230].…”

“…It should be noted that a UV filter assembly is necessary due to absorbance of most ampholytes at low-UV wavelengths. Native fluorescence is an alternative [211], if the instrument supports the detection mode and offers improved sensitivity [192]. Capacitively-coupled contactless conductivity detection (C 4 D) detection (Section 7.1) in combination with microchip CIEF has been recently reported.…”

“…Urea is still one of the most used additives to prevent precipitation [192, 209–211, 216, 219, 223, 224, 267, 268]. However, several other substances have proven their usefulness.…”

Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection

“…CZE is, among CE-SDS, capillary isoelectric focusing (CIEF), and imaged capillary isoelectric focusing (iCIEF), one of the routinely used electrophoretic techniques for the analysis of biopharmaceuticals in general and mAbs in particular [21][22][23]. While the detection of mAbs through induced fluorescence is described in combination with CE-SDS [18,24,25] and iCIEF [26,27], its combination with CZE has not been explored to date and is now being investigated for the first time. Since the labelling of mAbs with fluorescent dyes would change their charge-to-size ratio, native fluorescence should be favored so as to not impair the separation.…”