Scale-up Production and Immunological Assessment of Recombinant SARS- CoV-2 Spike RBD in Pichia pastoris

With more than two years in COVID-19 pandemic, the underlying virus is mutating which indicates it is not over yet hence SARS-CoV-2 Spike RBD is a potent candidate for immunological development purposes. Here, we produced a recombinant-RBD protein from micro- to macro-scale production by Pichia pastoris with high purity that was assessed by immunological tests. A recombinant-RBD compromising 283 residues (31kDa) was constructed after epitope analyses. The target gene was initially cloned into Escherichia coli Top10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. The production was scaled-up in a 10L fermenter after 1L shake-flask cultivation. The product was ultrafiltered and purified using ION-Exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by ELISA test to evaluate the antigenicity and specific binding of the produced protein. Bioreactor cultivation yielded 4g/L of target protein after 160 hours fermentation, and ion-exchange chromatography indicated a purity of > 95%. Human serum ELISA test performed in four parts (1–4) and ROC curve area under curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was (100, 91.5) respectively. In conclusion, the recombinant-RBD can be used for IgG-based serologic kit and preventive purposes for patients with COVID-19 infection.