A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10
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