Charge variant analysis is important in the characterization of monoclonal antibodies because it provides important information about product quality and stability. Charge variants can be modified by C-terminal lysine modification, N-terminal pyroglutamic acid formation, deamidation and oxidation, and post-translational modifications. Charge variants of biological products may have an impact on stability and biological function. Variability can be an inherent feature of a host cell line and it can be introduced by commercial manufacturing methods. ICH Q6b requires biological products to be understood and tested for charge variants.
cIEF is an orthogonal technique for assessing protein charge heterogeneity. The charge variants are separated by their difference in isoelectric point (pI), which are separated when they migrate within the pH gradient under the influence of the applied electric field during the focusing step. The acidic, predominant and basic peak regions of pI are determined based on known pI markers.
IEX-HPLC separates proteins based primarily on the heterogeneity of surface charge; however, it may also be affected by structural heterogeneity and other modifications that affect the molecular interaction with the ion exchange resin. Therefore, the observed chromatographic analysis may be caused by a combination of charge differences and protein structural variants. Characterization of the acidic and basic peak fractions can be accomplished by fractions and then by peptide mapping and other orthogonal analysis techniques.
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