Optimization of an IgG1 CIEF separation by using narrow-range ampholytes and DMSO as protein solubilizer

CIEF is a powerful separation tool utilized in the characterization and relative quantitation of therapeutic mAb charged isoforms. However, one CIEF method is not capable of separating all mAbs with high resolution and reproducibility. Optimization of sample composition and separation parameters is expected when developing a CIEF method for a specific mAb. This paper summarizes a root cause investigation into why a validated CIEF separation method for MAK33 (a type of IgG1) was no longer reproducible. In addition, this paper introduces the concept of sample focusing volume, which is defined as the actual capillary volume occupied by the sample after focusing and explains why there is less protein precipitation and aggregation when using narrow-range ampholytes than broad-range ampholytes. The use of DMSO as protein solubilizer and possible replacement of urea is also explored in this work. Finally, this paper demonstrates that a new optimized CIEF method can achieve over 100 reproducible high-resolution separations of MAK33 per neutral-coated capillary.