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

Abstract

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high-resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis-Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis-Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε-amino-caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico-chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5-15% and 15-30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.

Stepwise optimization and sensitivity improvement of green micellar electrokinetic chromatography method to simultaneously determine some fluoroquinolones and glucocorticoids present in various binary ophthalmic formulations

“…Methanol serves as an EOF marker to monitor the changes in migration time, indicating variations in the driving power of EOF. Also, the choice of alkaline pH rather than neutral or acidic was based on the fact that the alkaline medium kept the inner silica of the capillary tube ionized (Goyon et al, 2018), thus minimizing the run time. The observed increase of apparent mobilities with increasing the buffer pH was attributed to a predominant effect of pH on EOF.…”

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

“…CE possesses many advantages, including fast separation, small sample consumption and high resolution, and can be considered as a key complementary technique to chromatographic approaches for the analysis of protein biopharmaceuticals [53,70,71]. The different electrophoretic modes that can be employed, such as capillary gel electrophoresis (CE-SDS) [72], capillary isoelectric focusing (cIEF), imaged cIEF (icIEF) [73] and capillary zone electrophoresis (CZE) [74][75][76], exhibit attractive opportunities for the characterization of ADCs at different levels, such as intact or reduced charge-or size-variants, isoforms, glyco-profiling and PTMs [26,70,71].…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…The majority of detailed protocols in the literature concerns the use of a dynamic HPMC coated capillary which is considered as a reference for mAb charge variants analysis [14,83,84]. However, different strategies have already been described with other dynamic positive coating as Polybrene [85] or neutral coating as polyethylene oxide [86,87]. Apart from this parameter, the optimization of experimental conditions concerning the mAb analysis in CZE depends enormously on the physicochemical properties of the mAbs to characterize them at the desired levels and will be described in the application section 4.2. asparagine sequence variation sites on the mAbs.…”

“…Described work is promising for a successful development of CZE as a major method for biopharmaceuticals analysis. Alternative approach consisting of exploring new BGE conditions for mAb charge variants separation, has also been developed by Goyon et al in 2018 [87]. 17 marketed mAbs have been evaluated using solely a Bis-Tris buffer (pH 7.0) and PEO dynamic coating.…”