Characterization of the acidic species of a monoclonal antibody using free flow electrophoresis fractionation and mass spectrometry

Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization. This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques. The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.