Analytical comparability of a plant-based biosimilar candidate for antitumoral antibody Pembrolizumab

Introduction: Pembrolizumab, an IgG4 antibody, is employed in immunotherapy of several cancer types.The reference product (RP), marketed as Keytruda (MSD), is costly, and biosimilars offer an opportunity for affordable access after patent expiration.Protein expression in plants represents an innovative solution towards the production of high-cost biologics at a lower price.Bio-Manguinhos (BM) is currently developing a biosimilar candidate for Pembrolizumab in plants (p'mAb).In line with regulatory requirements, biosimilars must demonstrate comparability for Critical Quality Attributes (CQA) with the RP through characterization methods.Objectives: Assess the analytical comparability between the biosimilar candidate developed by BM and the RP through different methods.Methodology: For structural characterization, UV spectroscopy, circular dichroism (CD), tryptophan fluorescence, isoelectric point determination by IEF-PAGE, thermal stability evaluation by NanoDSF, free thiol groups quantification by Ellman's assay, and homogeneity and relative molecular weight analysis by size exclusion (SEC) and ion exchange (CEX) chromatography, were performed.Binding affinity measurement to the PD-1 receptor was evaluated by microscale thermophoresis (MST).All assays were conducted simultaneously for both the reference product and the biosimilar candidate. Results:The UV spectra of the biosimilar candidate were similar to the RP, CD showed secondary structures predominantly composed of regular beta-sheets.Tertiary structures were also comparable, showing maximum fluorescence intensity at 330nm for both p'mAb and Keytruda.IEF-PAGE analysis revealed three isoforms with pI values ranging from 7.66 to 7.78 for both molecules.Thermal kinetics showed melting temperatures of 68.9C for p'mAb and 63.8C for Keytruda.PD-1 binding assays indicated comparable affinity (kDs: 1.37 -3.17 M for Keytruda and 1.85 -3.34 M for p'mAb).Ellman's assay revealed free-sulfhydryl groups were not detected.The estimated molecular weight for both were approximately 150kDa, as determined by SEC analysis.Additionally, the CEX effectively distinguished potential molecular variants. Conclusion:All methods enabled structural characterization of the analyzed CQA's from both molecules with promising biosimilarity results.Differences must be evaluated through additional, orthogonal methods.The establishment of these methods are important tools to support the R&D of biosimilar products.