2.2. Capillary Iso-Electric Focusing (cIEF)

Capillary Isoelectric Focusing utilizing the iCE3 platform (IEF, PrinCE micro injector auto sampler, iCE 3 software package v. 3.0 and Chrom Perfect iCE3analysis software v. 6.0.4; ProteinSimple, San Jose, CA, USA) is an assay used to evaluate the charge heterogeneity of charged isoforms of proteins. cIEF is a charge-based separation technique that separates molecules based on their isoelectric point (pI). Samples are prepared by mixing the protein of interest with selected carrier ampholytes and pI markers. Then, the samples are loaded into the capillary cartridge (cIEF cartridge FC-coated (ProteinSimple, San Jose, CA, USA, Cat # 101701). Acid and base are added to the electrolyte tanks on the cartridge, and a voltage is applied in which analytes are focused at their pI. The focusing step is captured in real time by a charge-coupled device (CCD) camera, which takes a picture of the entire length of the capillary column every 30 s. The resulting separation is an electropherogram that identifies the pI and absorbance of the protein peaks. The samples comprised 50 µL of 2 mg/mL mAb, 100 µL of master mix solution, 150 µL of ultra-pure water. Each single mL of master mix solution comprised 740 µL of 1% Methyl Cellulose Solution, 200 µL of 8–10.5 ampholyte, and 20 µL each of 3–10 ampholyte, pI standard 7.9, and pI standard 10.1. The focusing times in focus periods 1 and 2 were 3.0 and 10.0 min, respectively, while the voltages in focus periods 1 and 2 were 500 and 3000 V, respectively.