Molecular pharming for production of biosimilar Pembrolizumab in plants: codevelopment of an anti-PD-1 monoclonal antibody

Introduction:Pembrolizumab is an anti-PD-1 monoclonal antibody widely used for different tumor indication.A biosimilar aims to replicate the reference product (RP) characteristics, with minimal variations, while ensuring safety and efficacy parameters.Plant expression systems offers benefits such as lower costs compared to mammalian cell cultures.This study proposes the production of a Pembrolizumab biosimilar using Nicotiana benthamiana as a non-conventional expression platform.Objectives: To develop an alternative efficient method for producing a Pembrolizumab biosimilar in N. benthamiana at the Bio-Manguinhos lab in Ceara.Methodology: Plants were cultivated in a growth chamber at 24C, with a 16h/8h light/dark photoperiod, light intensity of 200 molm -s -, and 80% humidity.After 46 days of cultivation, the plants were vacuum-infiltrated with recombinant Agrobacterium tumefaciens suspension for transient expression of Pembrolizumab and cultivated for seven days.The crude extract was obtained and clarified by centrifugation, followed by filtration in 0.22 m.Purification included affinity chromatography, strong cation exchange, and anion exchange chromatography.SDS-PAGE and Western blotting analyses were conducted to compare the plant-produced Pembrolizumab with the RP.Quantification and kinetics (dissociation constant assessment) were performed via Bio-Layer Interferometry (Octet).Results: From 222 g of biomass, 11 mg of biosimilar Pembrolizumab were obtained.After purification, 8 mg of protein were recovered, yielding 36 mg of protein per kilogram of plant biomass.SDS-PAGE and Western blotting confirmed a MW profile similar to RP Keytruda , and kinetic analysis showed comparable KD values and affinity constants in the pM range, as displayed in the literature. Conclusion:This study highlights the potential of this N. benthamiana based expression system for the efficient production of biosimilar monoclonal antibodies.The obtained antibody demonstrated kinetic data and affinity constants comparable to those of Keytruda , suggesting molecular similarity.However, more data is needed to confirm similarity and upstream optimization is required to enhance biosimilar yield.