17 Instrument
iCIEF was performed using a CEInfinite Analytical iCIEF instrument equipped with an energy-efficient LED UV (280 nm) light source and scientific CMOS camera, eliminating the need for external temperature control for the separation cartridge.
The autosampler sample storage temperature was kept at 10ºC.
25 µL of premixed mAb and IEF solution was injected into the iCIEF cartridge.
On-Board Mixing Figure 2: iCIEF Peak Integration with Clarity for Premixed and On-Board Mixed
(PAP), is more consistent with automation as variation introduced during manual mixing is circumvented.
Experimental
Imaged Capillary Isoelectric Focusing (iCIEF)
Materials
All chemical compounds were obtained from Advanced Electrophoresis Solutions Ltd. (AES, Cambridge, Ontario, Canada) unless otherwise specified.
The monoclonal antibody bevacizumab, or ‘mAb’, was received as a gift from Tuopuda Technology Co, Ltd (Beijing, China).
Solutions for iCIEF
Desalting of the mAb was not required. Undiluted mAb sample (10 mg/mL) was stored at 4ºC. All solutions were prepared with deionised water filtered with a Millipore MilliQ system.
Premixed mAb and IEF solution: 0.25 mg/mL of bevacizumab in 4.0% 3-10 HR AESlytes, 0.35% methyl cellulose, 0.50% 7.05 pI Marker, 0.50% 9.46 pI Marker, 3.00 M urea in deionised water.
mAb dilution for on-board mixing: 2.5 mg/mL of bevacizumab in deionised water.
IEF solution for on-board mixing: 4.4% 3-10 HR AESlytes, 0.39% methyl cellulose, 0.55% 7.05 pI Marker, 0.55% 9.46 pI Marker, 3.33 M urea in deionised water.
45 µL of IEF solution for on-board mixing was aspirated from a glass vial into the autosampler needle. This fluid was then dispensed into a well in a 96-well plate containing 5 µL of 2.5 mg/mL mAb dilution for on-board mixing. The autosampler needle aspirated and expelled the solution twice to mix for a final mAb concentration of 0.25 mg/mL. Immediately after mixing, 25 µL of the total solution was injected into the iCIEF cartridge. Each injection was from an individual mixing event.
iCIEF Capillary Cartridge
Both the premixed and on-board mixed solutions were focused using the same CEInfinite WCID 100 µm ID fluorocarbon- coated (FC) cartridge. All standard CEInfinite WCID cartridges have a 5 cm separation capillary.
Focusing Conditions
The anolyte was 80 mM phosphoric acid and the catholyte was 100 mM sodium hydroxide. Both solutions were prepared in 0.1% methyl cellulose.
Sample was focused for 1.0 minute at 1.5 kV and then for 7.5 minutes at 3.0 kV. Focused profiles were imaged with 280 nm UV light.
Software
Electropherogram, or charge profile, data was exported from CEInsight software, the operating software for CEInfinite iCIEF instruments.
Figure 3: Six iCIEF mAb Electropherograms of Premixed 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.
pI and PAP values were calculated using Clarity (DataApex, Prague, The Czech Republic). For peak integration, the integration interval was ~7.6 to ~8.7 pI, the Global Peak Width was set to 0.100 pI, and the Global Threshold was 0.1000
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