Optimization of the Imaged cIEF Method for Monitoring the Charge Heterogeneity of Antibody-Maytansine Conjugate

Abstract

The aim of this study was to develop a whole-column imaging-detection capillary isoelectric focusing (icIEF) method for the analytical characterization of charge heterogeneity of a novel humanized anti-EphA2 antibody conjugated to a maytansine derivative. In addition to focusing time, sample composition was optimized: pH range, percent of carrier ampholytes, conjugated antibody concentration, and urea concentration. A good separation of charge isoforms was obtained with 4% carrier ampholytes of a large (3-10) and narrow pH range (8-10.5) (1 : 1 ratio), conjugated antibody concentration (0.3-1 mg/ml) with a good linearity (R²: 0.9905), 2 M of urea concentration, and 12 minute for focusing. The optimized icIEF method demonstrated a good interday repeatability with RSD values: <1% (pI), <8% (% peak area), and 7% (total peak areas). The optimized icIEF was useful as an analytical characterization tool to assess the charged isoform profile of a discovery batch of the studied maytansinoid-antibody conjugate in comparison to its naked antibody. It exhibited a large pI range (7.5-9.0), while its naked antibody showed a narrow pI range (8.9-9.0). In the discovery batch of maytansinoid-antibody conjugate, 2% of charge isoforms had the same pI as the pI of naked antibody isoforms.

Figure 1

Analysis of maytansinoid-antibody by icIEF. Experimental conditions: final concentration 1 mg/mL in 0.35% methyl cellulose, 2% pharmalytes 3–10 and 2% pharmalytes 8–10.5 (1 : 1 ratio), 2 M urea, pI markers: 6.61, 9.50. Focusing time: 10 min at 3000 V. λ = 280 nm.

Figure 2

Analysis of different concentrations of maytansinoid-antibody by icIEF (0.3, 0.5, 0.8, 1, and 1.5 mg/ml). Other experimental conditions are as mentioned in Figure 1.

Figure 3

Plot of total peak area against maytansinoid-antibody concentration (0.3, 0.5, 0.8, 1, and 1.5 mg/ml).

Figure 4

Effects of urea concentration on the separation of charge isoforms of maytansinoid-antibody by icIEF (0, 1, 2, and 3 M urea). Other experimental conditions are as mentioned in Figure 1.

Figure 5

Effect of urea concentration on the pI values of main ADC charge variants.

Figure 6

Effects of focusing time on the separation of charge isoforms of maytansinoid-antibody by icIEF. Other experimental conditions are as mentioned in Figure 1.

Figure 7

Effects of % pharmalytes on the separation of charge isoforms of maytansinoid-antibody by icIEF. Other experimental conditions are as mentioned in Figure 1.

Figure 8

icIEF profiles of naked antibody (a) and its corresponding maytansinoid conjugate (b). Final concentration of maytansinoid-antibody in sample matrix is 1 mg/ml diluted in 0.35% methyl cellulose, 4% 3–10 pharmalytes/8–10.5 pharmalytes (1 : 1 ratio), and 2 M urea, pI markers: 8. 18, 9.50 (a) and 6.61, 9.50 (b), and focusing time of 12 min at 3000 V. Other experimental conditions are as mentioned in Figure 1.