18 February / March 2019


protein samples when compared to manual premixing. For method transfer to this more efficient iCIEF process, there must be a negligible loss of precision in charge heterogeneity profile data. Both the premixing and on-board mixing options for the CEInfinite Analytical instrument were used to measure equivalent solutions of bevacizumab to compare reproducibility, as measured by % RSD. All values for the premixed are in Table 1 and those for the on-board mixed can be found in Table 2.


The results obtained using the CEInfinite instrument will also be compared to a study by Sosic et al. published in Electrophoresis that utilised a comparable iCIEF instrument to study a very similar mAb as evidenced by its peak pI and PAP values [10].


Figure 4: Six iCIEF mAb Electropherograms of On-Board Mixed mAb. Major peaks are marked with the associated charge variant label. mAb concentration for all injections was 0.25 mg/mL. Not shown are the 7.05


and 9.46 pI markers.


mAbsorbance. This yielded profiles similar to those in Figure 2 for each mixing method.


The data was tabulated and analysed in Excel (Microsoft, Redmond, WA).


Results


As a baseline for comparison, the mAb was manually prepared to a final concentration of 0.25 mg/mL in IEF solution. To quantify the repeatability of the mAb charge profile, injection of this sample was repeated five times, for six injections total. These six profiles are overlaid in Figure 3 to show the consistency between the injections. The four dominant charge variants of the mAb are labelled in Figure 3: the minor


and major acidic peaks, acidic 1 and acidic 2, respectively; the main peak; and the basic peak.


To quantify profile consistency, the percent relative standard deviation (% RSD) of both the pI and the PAP was calculated for each


of the four charge variants and is displayed in Table 1.


To compare the precision of on-board mixing to manual premixing, the mAb stock was diluted to 2.5 mg/mL, diluted ten-fold in IEF solution, and mixed promptly before injection into the cartridge separation capillary. The mixing and injection process was repeated five times from a total of six different sample wells. These on-board mixed charge profiles are layered in Figure 4 to highlight their consistency across mixing events.


The pI and PAP of the four major charge variant peaks were determined to evaluate the precision of the on-board mixing function. The results are summarised in Table 2.


Discussion


The automated nature of on-board mixing saves time in the preparation of individual


Table 1: Total pI and PAP Data from iCIEF of Premixed mAb. The reproducibility of iCIEF separation was determined by % RSD for both pI and PAP for the four main charge variants of bevacizumab.


When comparing variation in the pI values for the main mAb peak, the premixed was slightly greater than that seen in on-board mixing (0.03% vs 0.02%). In the Electrophoresis study with the comparable mAb, the main peak pI % RSD was three- fold greater than seen from the CEInfinite (0.1% RSD) [10]. For variation in pI for the main acidic peak, acidic 2, the pI precision was similar between the premixed and the on-board mixed (0.04% vs. 0.05% RSD). This does not outperform the comparable instrument, as the Electrophoresis study reported 0.0% RSD for the main acidic peak [10]. For the minor acidic peak, acidic 1, the pI RSD for the premixed was lower than the on-board mixed (0.02% vs. 0.13% RSD). The earlier work did not calculate the pI for the minor acidic peak [10]. The premixed was outdone by on-board mixing for the basic peak pI (0.11% vs. 0.08% RSD), which was not calculated in Sosic et al. [10].


The % RSD value for the main peak PAPs was 45% higher for the premixed than the on-board mixed on the CEInfinite (1.15% vs. 0.79% RSD), and the RSD for the same peak in Sosic et al. was more than double the CEInfinite values (2.6% RSD) [10]. For the larger of the two acidic charge variant peaks, acidic 2, on-board mixing was over twice as precise when comparing PAP than premixing (1.30% vs. 3.40% RSD). For acidic 1, the minor acidic peak, the % RSD values for PAP where very similar between methods (8.11% vs 8.34% RSD). In the previous study, the PAP for both acidic species were combined to yield an RSD of 6.9% [10]. The on-board mixed % RSD values for basic peak PAP were slightly lower than those for the premixed (10.10% vs. 10.66% RSD) and both values outperformed when compared to the previous Electrophoresis study (17.1% RSD) [10].


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