Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused‐silica capillary

Abstract

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

“…In the obtained electropherograms of ( Figure 1b) the basic and acidic variants can be observed at the sides of the main component, Rtx. In general, the acidic variants are formed by glycosylation with sialic acid or uronic acid, or by deamidation of asparagine or glutamine, or by pyroglutamate formation from glutamine and glutamate, whereas the basic variants are formed by formation of Fc-1 lysine or Fc-2 lysine, or by the noncyclization of N-terminal glutamine, or by succinimide formation from aspartic acid polyamins, polymers) added to the running electrolyte have been investigated as dynamic coatings on the surface of the capillary [11][12][13][14][15][16]. Often a mixture of different additives with complex mechanism are applied [11][12][13][14][15][16].…”

“…However, the excess of TETA can modify the charge heterogeneity of mAb caused by the interaction of amines of TETA with the carboxyl groups of the mAb. The CZE separations of Rtx shown in Figure 2 were performed using running buffer containing 400 mM EACA, 2 mM TETA and 0.05% HPMC (suggested concentrations from [12] for several mAbs but not Rtx) of different pH values. At pH= 6.8 (close to the pI of Rtx) the adsorption of the Rtx to the inner wall is still considerable, which resulted in peak tailing (Figure 2a,b).…”

“…A little better resolution was obtained for a slightly larger electric field and longer separation length (Figure 3e), but the further increasement of the length of the capillary deteriorated the efficiency of the separation. Interestingly, the increase of the EACA concentration in the running buffer improved the resolution only for the basic variants for other mAbs [12].…”

“…This can often suggest modifying the inner wall of the capillary for preventing protein adsorption. Using bare fused silica capillaries different additives like 6-amino caproic acid (EACA) [11], triethylene tetramine (TETA) [12] or hydroxypropyl methylcellulose (HPMC) [11] are given into the background electrolyte. Shi et al evaluated the separation of charge variants of mAb in fused silica capillary using a CZE running buffer containing polyethylene oxide (PEO) and TETA [13].…”

Development and validation of a rapid capillary zone electrophoresis method for determining charge variants of mAb

“…Later Ma and Nashabeh [19] developed capillary zone electrophoresis (CZE) as a tool for the analysis of charge heterogeneity of therapeutic mAbs in a permanently coated capillary to minimize protein adsorption to the inner wall of the capillary. Yan [1,20] developed and optimized the CZE method in dynamically coated fused silica capillary to reduce separation time and cost.…”