15


sample concentrating technique iCIEF is the most powerful unequivocal molecular weight structure characterisation method when used in tandem with mass spectrometry (iCIEF-MS). It enables high resolution of pI-based separations and the MS identification of individual peaks. Neusüß’s group at Aalen University in Germany has demonstrated a two-dimensional system using iCIEF instrumentation by transferring charge variant peak individually via a nanolitre valve, coupled with CZE-MS for the MS protein charge variant structure characterisation [6]. Direct online CIEF–MS analysis of intact mAbs have been reported using either stainless-steel flow-through micro vial interface and a pulled-glass electrokinetically pumped sheath-flow interface [7-11].


In this report, direct coupling iCIEF to Thermo Fisher Scientific Obitrap QE MS with low flow HPLC ESI source will be demonstrated.


Experimental Materials


All chemical compounds were obtained from Advanced Electrophoresis Solutions Ltd (AES, Cambridge, Ontario, Canada) unless otherwise specified.


Monoclonal antibody NISTmAb was Purchased from Millipore Sigma (cat. no. NIST8671).


Solutions for iCIEF


Desalting of the NISTmAb was not required. Undiluted NISTmAb sample (10 mg/mL) was stored at 4°C. All solutions were prepared with deionised water filtered with a Millipore MilliQ system.


For analytical iCIEF: 0.25 mg/mL of NISTmab in 1% HR 3-10 and 3% HR 8-10.5 AESlytes, 0.35% methyl cellulose, 0.50% 8.18 pI Marker, 0.50% 9.77 pI Marker, 2.00 M urea is mixed in deionised water.


For prep iCIEF: 2 mg/mL of NISTmab in 1% HR 3-10 and 3% HR 8-10.5 AESlytes, 0.50% pI Marker 8.18, 0.50% pI Marker 9.77, 2.00 M urea is mixed in deionised water. Mobilisation solution: 10mM Iminodiacetic acid; make up solution: distilled water.


For iCIEF-MS: 1 mg/mL of NISTmab in 1% HR 3-10, 3% HR 8-10.5 AESlytes, and 20% formamide are mixed in deionised water.


Instrumentation


Preparative imaged Capillary Isoelectric Focusing (iCIEF) was performed using a


Figure 2: Illustration of highly efficient prep iCIEF and automated iCIEF fractionations.


Figure 1: CEInfinite multi-functional iCIEF platform.


CEInfinite multi-functional system equipped with an energy-efficient LED UV (280 nm) light source and a CMOS camera.


The anolyte was 80 mM phosphoric acid and the catholyte was 100 mM sodium hydroxide. Both solutions were prepared in 0.1% methyl cellulose. The autosampler sample storage temperature was kept at 10°C.


Separation capillaries for iCIEF


WCID cartridge, 100 µm ID, fluoro carbon (FC) coated capillaries is used for all analytical iCIEF. 200 µm ID acrylamide coated (AD) capillary cartridges (AES, cat. no. CP00303) and microtee integrated (AES, cat. no. CP00303M) are used for iCIEF-MS; 320 µm ID AD coated cartridges (AES, cat. no. CP00307) are used for preparative iCIEF. All these WCID cartridges have a 5 cm long separation capillary, and 50 µm ID transfer capillary are assembled for both iCIEF-MS and preparative iCIEF cartridges. The 200 µm AD coated iCIEF-MS cartridge used for iCIEF-MS includes a quartz union (works as a microtee), connecting the make- up solution and transfer capillary to ESI of MS. Both the make-up solution capillary and transfer capillary have a 100µm ID.


iCIEF-MS


A Thermo Exactive Plus EMR mass spectrometer equipped with an Ion Max ESI Ion Source with a 34-gauge needle (Thermo


Fisher Scientific, Bremen, Germany) was used for mass measurement. The spray voltage: 3.8 kV, sheath gas: 20 psi, Auxiliary gas: 0, Probe heater: 80°C, Capillary temp: 320°C.


The focusing was 1 min at 1000 V, 1 min at 2000 V and 7 min at 3000 V, and 3000 V during mobilisation; the mobilisation speed was 0.05 µL/min across the separation capillary, and 5 µL/min make up solution added through a micro tee. Mobilisation time was 15 min.


Software


The control and data acquisition of both analytical and prep is with CEInsight, which is CFR21 part 11 compliant. Charge profile data was exported from CEInsight and processed with Clarity (DataApex, Prague, The Czech Republic).


Principle of Preparative iCIEF


Capillary isoelectric focusing is a two-step process. After introducing the sample, 22 µL of premixed solution of the mAb, ampholytes and optional additives, into the separation capillary, the voltage is applied. The sample solution is focused for 1.0 minutes at 1.5 kV and then for 7.5 minutes at 3.0 kV. With the CEInfinite instrument, the focusing process can be monitored live in 15-second intervals. The focusing process is illustrated in the top graphic of Figure 2.


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