In vitro
This study describes the development of a high-yield Chinese hamster ovary (CHO) cell manufacturing platform for a biosimilar of dulaglutide, a GLP-1/IgG4-Fc fusion protein approved for type 2 diabetes. Researchers used apoptosis-resistant CHO 4BGD cells and sequentially transfected two expression plasmids encoding dulaglutide, employing a two-step transgene amplification strategy using methotrexate (MTX) followed by methionine sulfoximine (MSX) selection. The dual-selection approach resulted in approximately 30% higher titers in polyclonal populations compared to MTX amplification alone. Through a clonal cell line selection pipeline, the top clone (4BGD/Dul #73) achieved a product titer of 1.05 g/L in a 3-week fed-batch process, with a specific productivity of up to 22 pg·cell⁻¹·day⁻¹ and stable expression over 69 days without selective pressure. Purity assessed by size-exclusion HPLC showed ≥95% monomer content. Biological activity testing in a GLP-1 receptor/CRE-luciferase reporter assay yielded an EC₅₀ of 52 pM for the biosimilar candidate versus 76 pM for the reference drug. Limitations include the absence of in vivo or clinical data, with all findings limited to cell culture and in vitro bioassay systems.
Pharmaceuticals (Basel, Switzerland) · Dec 2025DOI ↗ In vitro
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.
International journal of biological macromolecules · Sep 2025DOI ↗