CE-SDS method as a tool for developing biosimilars

Introduction: Monoclonal antibodies have been a revolution in therapeutics medicine, with rapidly growing research and development, and a market poised to grow at a compound annual growth rate of 11.07% from 2024 to 2033.Biosimilars have also grown, with 68 biosimilars approved by the FDA until 2024.To produce a Biosimilar it is necessary to demonstrate high similarity to the reference product in terms of purity, chemical identity, and bioactivity.CE-SDS is a sensitive analytical method for estimating Low Molecular Weight Impurities such as Non-Glycosylated and Antibody fragments, which is used for characterization and stability assessment.Objectives: Implementation of a CE-SDS method to support the development of Biosimilar molecules of Bio-Manguinhos.Methodology: Analyses were performed using a bare-fused silica capillary with 50 m i.d. and 67 cm total length, with a 100 mM Tris-HCl (pH 6.8 ) buffer and 1% SDS.A 10 kDa Internal Standard was added to the protein samples, along with 2-mercaptoethanol to reduced conditions and Iodoacetamide to non-reduced conditions.0,1M HCL and 0,1M NaOH were used to capillary condition/regenerate between injections.Separation was conducted by a 300 A and detection was performed by a Photo Array Diode (PDA) at 220 nm.Commercially available mAbs related to pembrolizumab nivolumab were analyzed, and compared to Bio-Manguinhos Biosimilars.Results: Commercially available mAbs demonstrated viability for the method conditions, with reproducibility between injections and different batches.Commercially available mAb related to pembrolizumab has a purity in Non-reduced conditions above 96% and commercially available mAb related to nivolumab above above 92%.In Reduced conditions, all commercial mAbs obtain results above 99% (considering Light Chain+ Heavy Chain+ Non-Glycosylated Heavy Chain).Biosimilars in development presented similar migration time of Light Chain, Heavy Chain, Non-Glycosylated Heavy Chain (Reduced conditions), and Intact IgG (Non-Reduced conditions) when compared to their respective reference products, but with lower purity level, and a higher presence of antibody fragments, indicating potential differences in product stability and process consistency. Conclusion:The implemented CE-SDS method proved to be sensitive and robust enough to be applied to monoclonal antibodies and become a powerful tool for Bio-Manguinhos Biosimilars development.