Multiple analysis based on dual-mode anion-exchange chromatography strategy reveals significant impact of charge heterogeneity on structure and function of dulaglutide.
This study developed a dual-mode semi-preparative anion-exchange chromatography (AEX) method to fractionate and characterize charge variants of dulaglutide, a GLP-1 receptor agonist used in type 2 diabetes management. Because dulaglutide is an acidic Fc-fusion protein with complex charge heterogeneity, standard characterization methods are technically challenging. The researchers isolated acidic, main, and basic charge variant fractions and subjected them to comprehensive downstream analyses, including assessments of sialic acid content, post-translational modifications (phosphorylation, sialylation, deamidation, oxidation), size heterogeneity, aggregation, truncation, and biological activity. A key finding was that aggregates in basic variants are primarily held together by non-covalent interactions, while acidic variants contain covalently linked aggregates—a structurally meaningful distinction. Charge variants showed only slight differences in biological activity, potentially linked to aggregate presence. A comparative analysis between the innovator product Trulicity® and a biosimilar candidate revealed minor differences in acidic variants, likely attributable to variations in phosphorylation and sialylation profiles. Limitations include the in vitro nature of the biological activity assessments and the absence of in vivo or clinical data. The study provides a detailed analytical framework for characterizing charge heterogeneity in complex biopharmaceuticals.
Why this grade: The study is entirely analytical and in vitro in nature, involving chromatographic fractionation and cell-based/biochemical assays with no human subjects or animal models.
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.
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