Characterization of antibody charge heterogeneity resolved by preparative immobilized pH gradients.

A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures. More recently, an off-line method that uses pI-based separation on immobilized pH gradients was developed to preparatively isolate material with convenient liquid phase recovery. This method uses the Agilent OFFGEL 3100 Fractionator and was optimized to produce fractions of antibody charge isoforms differing by as little as 0.1 pI units. The isolation of highly resolved fractions then allowed for the identification of N- and C-terminal basic charge modifications including noncyclized glutamine, signal peptide extensions, and various levels of C-terminal lysine processing and high mannose structures. These species could then be correlated to specific peaks in the cIEF profile. This work shows that a preparative IEF method using immobilized pH gradients can be optimized to generate highly resolved, pI-based fractions in solution which can be used for successful cIEF profile characterization. Access to preparative amounts of discrete charged species allows for a better understanding of the underlying covalent modifications responsible for the charge differences and facilitates evaluation of the impact of these modifications on stability and potency of therapeutic antibodies.