5
Results and Discussion Adjustment of CA concentration and focusing conditions
This study tested the separation performance of cIEF with different brands of CAs, with a mAb sample having multiple charge isoforms with isoelectric points between 6 and 7. To ensure a fair comparison, the concentration of every CA brand was adjusted according to two criteria:
(i) migration of pl marker 5.5 and 7.0 within a time window of 25 to 33 minutes, and
(ii) a 3.5 to 4 minute migration time difference between these two pl markers (data not shown).
Optimised concentrations were 4.7% v/v Pharmalyte and HR AESlyte (6 µL stock solution added to the sample solution), 9.0% v/v SH AESlyte (12µL), and 1.3% w/v Servalyt (4 µL; this CA brand was supplied as a 40% w/v stock solution). With these relatively high concentrations of Pharmalyte, HR and SH AESlyte the most acidic part of the pH gradient couldn’t be observed, leading to a frequent loss of pl marker 4.1 (data not shown). If this part of the pH gradient is of interest, lower CA concentrations have to be employed. The focusing time used was 10 minutes, except for Servalyt, where the focusing time had to be increased to 12 minutes to get complete focusing of all pl markers (data not shown).
Carrier ampholyte specifi c background
The CA specifi c background was monitored by cIEF runs with injection of pl markers only (Figure 1). The lowest background was observed with Pharmalyte and HR AESlyte, and a slightly enhanced background with SH AESlyte. A signifi cantly enhanced background was seen with Servalyt. In line with this observation, diluted stock solutions of Servalyt showed, in comparison to the other CA brands, a higher absorbance at 280 nm (data not shown).
Figure 2. CIEF analysis of the mAb test sample with different CA brands. Shown is the range between pl marker 7.0 and 5.5 of representative electropherograms obtained with Servalyt, SH AESlyte, HR AESlyte and Pharmalyte (from top to bottom). For better comparability, the time axis of the electropherograms were aligned with both pl markers as reference points (indicated by the red vertical lines). Isoform peaks that were automatically integrated are consecutively numbered from basic to acidic. Integration limits are indicated by red dotted lines.
Table 1. Intermediate precision of apparent pI measured with different CA brands. For each CA brand sets of 6 runs were done on 3 different days and with 3 capillary batches (n=18). For the assignment of isoform peaks refer to Figure 2.
Isoform Pharmalyte Average RSD%
Figure 1. Background obtained with different CA brands. CIEF runs were done with sample solution without mAb test sample. Shown is a zoom in on the baseline the range between pl marker 7.0 and 5.5 of representative electropherograms obtained with Servalyt, SH AESlyte, HR AESlyte and Pharmalyte (from top to bottom). For better comparability, the time axis of the electropherograms were aligned with both pl markers as reference points (indicated by the red vertical lines).
Resolution
Figure 2 shows electropherograms obtained for the mAb test sample with different CA brands. In comparison to the similar isoform pattern observed with Pharmalyte and HR AESlyte, the isoform pattern obtained with SH AESlyte and Servalyt was shifted by 0.10 to 0.15 pH units to the more acidic range. Seven isoforms could be clearly resolved, and automatically integrated with Pharmalyte and HR AESlyte. A slightly increased resolution was observed for SH AESlyte, with additional shoulders for peaks in the middle of the isoform pattern. However, a lower resolution was observed for the more acidic isoforms (SH AESlyte isoforms 6 and 7 in comparison to HR AESlyte and Pharmalyte isoforms 5 to 7, Figure 2). As for Pharmalyte and HR AESlyte, seven isoform peaks were automatically integrated with SH AESlyte (Figure 2). The highest resolution was obtained with Servalyt, that permitted identifi cation of nine isoform peaks. This CA brand produced an overall somewhat different isoform pattern in comparison to the other three CA brands (Figure 2). The higher resolution obtained with Servalyt, in comparison to Pharmalyte, is in agreement with the higher content of CA isoforms in the pH range 6 to 8 [3] by exploring, through a 3-D methodology (Rotofor fractionation followed by CE MS).
1 2 3 4 5 6 7 8 9
0.029 HR AESlyte Average RSD% SH AESlyte Average RSD% Servalyt Average RSD%
6.720 0.033 6.727 0.006 6.570 0.026 6.615 0.011 6.655
6.564 0.011 6.583 0.005 6.461 0.022 6.487 0.015 6.392 0.019 6.419
6.664 0.065 6.477 0.036 6.572 0.013 6.394 6.369
6.335 0.023 6.363 0.014 6.269 na na
na na
na na
0.011 6.282 6.198
na na
0.030 6.503 0.005 0.025 6.463 0.029
0.019 6.416 0.018 0.028 6.344 0.010
0.020 6.300 0.013 6.114 0.022 6.301 0.020 na na
na na
6.265 0.006 6.171 0.012
Intermediate Precision
The precision of the method including within-laboratory variations such as different day and capillary batch was determined for isoform pI and relative abundance of the mAb test sample (Tables 1 and 2). The intermediate precision for apparent pI obtained with all CA brands for all mAb isoforms was below 0.1 %RSD (Table 1). For relative peak area, the intermediate precision obtained with Pharmalyte and HR AESlyte was less than 3 %RSD for all isoforms (Table 2). With SH AESlyte and Servalyt, values less than 9 %RSD were observed. The only exception was with Servalyt, where the relatively small and poorly resolved isoform peak 7 showed an intermediate precision for relative peak area of 14 %RSD (Table 2 and Figure 2). The enhanced variability seen with SH AESlyte and Servalyt could be due to the more complex isoform pattern, making reliable integration more challenging (Figure 2), or the higher background observed with both CA brands (Figure 1). Overall, the precision results obtained with all tested CA brands agree well with published data obtained with a variety of different capillary coatings [4,6]. An interesting behaviour was observed with HR AESlyte for isoform peak 2, which showed a fourfold higher pI variability than any other peak with this CA brand (Table 1). In one set of measurements performed under repeatability conditions, this peak seems to switch between two shapes, one symmetrical and one with a small shoulder, as is observed with Pharmalyte (Figures 3 and 2), which might indicate some issues with sample stability.
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