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16 May / June 2021


capillary into the collection for charge variant peak is about 2-3 minutes, which allows the completion of charge variant fraction collection within 30 mins.


In Figure 3b the online coupling of iCIEF with ESI-MS is illustrated. A sheath solvent is added in short distance before the mass spectrometer entrance to optimise the conditions for electrospray ionisation.


Figure 3: a) Schematic of preparative iCIEF AFC system (top) and b) iCIEF-MS connection (bottom).


The AES patented capillary diameter transformation technique (CDTT) [4] allows fractionation of protein charge variant purely based on pI. Unlike capillary isoelectric focusing on a CE-instrument, which requires that the catholyte vial is exchanged with a low pH buffer to move the focused zones past the point of detection, in CEInfinite a precise syringe pump is used (indicated in Figure 2), which starts after completion of focusing stage. Mobilisation is performed at 0.16 µL/min for 30 min.


The charge variant peaks are pushed by the syringe pump from the inlet to the outlet of the separation column while maintaining the high voltage. By the continuous migration of hydroxide ions from the cathodic side and protons from the anodic side of the separation capillary, the charge variant peaks in the separation capillary remain focused under the electric field.


The inner diameter of the transfer capillary is much smaller than the separation column. Once exiting from the separation capillary, the charge variant zone length expands by


the inner diameter ratio square. For a prep cartridge with a separation capillary of 320 µm ID and a transfer capillary 50 µm, about 41 times peak expansion is expected. The peak expansion inside the transfer capillary, where no electric field is applied, effectively minimises unwanted remixing charge variant zones.


Automated iCIEF fractionation


Unattended automated sample analysis is highly desirable in protein separation and characterisation nowadays. Thanks to the CDTT, the charge variant peaks can be fractioned with high purity. Higher purity charge variant collections can be accomplished by heart cut fractionation. To facilitate collections and to improve fraction collection efficiency, automated fraction collection (AFC) with an integrated selection valve and micro tee (Figure 3a) was developed. A make-up liquid at 10-20x mobilisation flow rate was applied to minimise evaporation of the collected zone. The transfer time from the separation


To demonstrate the separation resolution and repeatability of NISTmAb iCIEF, an injection of this sample was repeated three times using a i.d 100 µm FC cartridge. The iCIEF profiles of the three runs are overlaid in Figure 4 to show the reproducibility of the iCIEF analysis of NISTmAb. The five dominant charge variants of mAb A are labeled in Figure 4: the first and second acidic peaks, acidic 1 and acidic 2, respectively; the main peak; and the major and minor basic peaks.


Mobilisation


The mobilisation speed has a significant impact on the efficiency and resolution of prep iCIEF, an elevated pressure and a mobilisation speed which is too high will cause disturbance of the focused protein iCIEF profile, while too low a pressure will reduce the efficiency of the prep iCIEF. The mobilisation speed was optimised and controlled so that overall resolution of protein was preserved.


Table 1: Summary of recommended mobilisation conditions (ID200, ID320).


Cartridge Part Number


CP00303M (ID200)


CP00307 (ID320)


Load Volume - Inject Volume


30-17ul 35-22ul


Mobilisations Conditions


25-45 min, 3000V 0.05-0.1ul/min


25-45 min, 3000V 0.12-0.20ul/min


To confirm the charge variants’ purity, the collected fractions were subjected to iCIEF analysis. 5 µL of the collected solution was mixed with 20 µL IEF solution with two pI markers. The top trace in Figure 5 shows the electropherogram of NISTmAb in the prep cartridge during the mobilisation step. The stack of iCIEF profiles for all fractions is shown in the lower trace. Table 2 lists the calculated purity of every variant.


Figure 4: Three repeated runs of NISTmAb. NISTmAb: 0.25 mg/mL; 1% AESlytes HR 3-10, 3% HR 8-10.5 AESlytes, 2 M urea, Focusing:1500 V (1 min) and 3000 V (8 min). Capillary i.d. 100 µm.


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