Multiple analysis based on dual-mode anion-exchange chromatography strategy reveals significant impact of charge...

Charge heterogeneity is a critical quality attribute in biopharmaceutical development, necessitating thorough characterization to ensure product quality, efficacy, and stability. Dulaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist consists of two GLP-1 analogs fused to a human IgG4-Fc fragment. Being an acidic Fc-fusion protein, investigating charge variants of dulaglutide poses significant challenges due to its complex charge heterogeneity, molecular instability, and technical constraints. A dual-mode semi-preparative anion-exchange chromatography method was developed to fractionate charge variants, providing insights into structural differences in charge heterogeneity and their potential impact on biological functions. This method offers high purity, low cost, large preparation volume, and wide instrument availability, while preserving protein structural integrity. Subsequent comprehensive characterizations with respect to charge heterogeneity, sialic acid, post-translational modifications, size heterogeneity, related substances and biological activities were conducted to enhance understanding of modifications related to microheterogeneity such as phosphorylation, sialylation, aggregation, truncation, deamidation, or oxidation, and to establish critical structure-activity relationship. Surprisingly, the aggregates in basic variants are primarily bound through non-covalent interactions, whereas covalently linked aggregates are present in acidic variants. Charge variants slightly affected biological activity potentially due to the presence of aggregates. The comparative study of the innovator product Trulicity® and biosimilar candidate revealed that minor differences in acidic variants were possibly attributed to variations in phosphorylation and sialylation. This study elucidated the structural origins and functional implications of dulaglutide's charge heterogeneity, offering promising and comprehensive solutions for fractionation and detailed characterization of charge heterogeneity in complex proteins.