Development of a cost-effective process for the heterologous production of SARS-CoV2spike receptor binding domain (RBD) using Pichia pastoris

CONSORCIO ARGENTINO ANTICOVID; RUBERTO L

Congreso; Pichia Congress 2021; 2021

Biogramatics and Research Corporation Technologies

The 2020 SARS-CoV2 pandemic demanded the development of suitable tools to face and manage the massive human infection. Viral proteins or its domain would be useful molecules to detect, treat, and prevent viral disease and its consequences. SARS-CoV2 belongs to the Coronavideae family. Coronaviruses are enveloped non-segmented positive-sense RNA viruses presenting four open reading frames (ORFs) for structural proteins -Spike, Envelope, Membrane, and Nucleocapsid. Spike complex mediates the viral and cellular membrane fusion by binding mainly to the angiotensin-converting enzyme 2 (ACE2) and RBD which is the interaction domain. RBD heterologous expression is useful for diagnosis purposes, as well as for immunization to obtain neutralizing antibodies or even a protein-based vaccine.The RBD coding sequence was optimized for its expression in the methylotrophic yeast P. pastoris. Sequence for Spike amino acid residues 319-537 was fused to a secretion signal (N-terminal) to direct the heterologous protein to the extracellular space. Additionally, a C-terminal Sortase-A recognition sequence and a His6 tag (C-terminal) were included. Transformed clones were selected, grown in BMG and induced in batch in BMM with methanol for 72 h. The clone with the highest expression level of RBD in the culture supernatant measured by SDS-PAGE was scaled up in a bioreactor. Thus, recombinant Pichia pastoris was cultured in a stirred tank bioreactor composed of a 7 L vessel and a control module (BioFlo 115, New Brunswick). Briefly, fermentations were carried out using 1.5 L of low salt medium (LSM) as previously described by Chen and co-worker. The bioreactor was seeded with 0.15 L of inoculum culture in BMG (OD600 17.5). Then, the initial cultivation volume was 1.65 L with OD600 of 1.6. For pH control, NH4OH 1,4 g/L and H3PO4 4.3 g/L were used. A four-stages process was applied. The first stage consisted of a batch culture using LSM medium with unlimited glycerol as carbon source (40 g/L) supplemented with 3.5 mL/L PTM1 and 3.5 mL/L biotin solution (0.02% w/v). Under these conditions yeast cells grew and reached a biomass level of 64 (OD600). In the second phase, a glycerol solution (600 g/L) was fed into the culture at a growth-limiting rate to increase cell concentration and gradually derepress the AOX1 promoter. To assure glycerol limitation, feeding was automatically regulated according to the percentage of dissolved oxygen (DO%) in the culture. When biomass reached a value of 152 (OD600), glycerol feeding was ended and the third stage, aiming inductor adaptation, was started by adding enough methanol to reach a concentration of 4 g/L. After 3,5 h a peak in dissolved oxygen (spike) was recorded, suggesting that methanol was completely consumed. Then the fourth stage, expression induction, was carried out by adding pure methanol in a fed-batch mode. Feeding at a growth limiting rate was automatically regulated with the level of DO % in the culture. This stage lasted for 108 h reaching a cell density of 332 (OD600). Total protein concentration in the supernatant was 1340 mg/L and RBD reached a concentration of 520 mg/L, representing 38% of supernatant total proteins. The final culture volume was 2.8 L containing 1456 mg of RBD. Whole process volumetric productivity was 3.8 mg RBD/L.h. Product (RBD)/biomass yield (YRBD/X) resulted in 5.8 mg RBD/g DCW, while RBD/methanol yield (YRBD/S) was 1.9 mg RBD/mg methanol. The whole process was carried out using only compressed air as the oxygen source. The use of a basal salt medium with simple carbon sources and the provision of oxygen only from air, combined with the obtained RBD concentration resulted in a process with a relatively low production cost. A simple downstream procedure was applied, using centrifugation, NTA-Ni affinity chromatography and dialysis. A 34% of the raw RBD amount in the culture supernatant was recovered, showing a purity >95% (RP-HPLC). The whole process represented a significant improvement in the RBD production (almost 5 times) compared to our first report (Argentinian AntiCovid Consortium, 2020).