Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate

Abstract

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.

Keywords: Pichia pastoris; circular dichroism; hydrophobic interaction chromatography; protein characterization; protein purification.

Figure 1

Purity assessment for the production process. SDS-PAGE with Coomassie Blue staining under (a) nonreduced condition and (b) reduced condition. M, SeeBlue plus 2 protein marker; lane 1, original fermentation supernatant (FS); lane 2, concentrated FS after tangential flow filtration (TFF); lane 3, butyl HP elution pool (HIC); lane 4, concentrated HIC pool (post-HIC TFF); and lane 5, the SEC pool/final purified RBD219-N1 (SEC) and (c) quantified purity at different purification steps.

Figure 2

Purity assessment of purified RBD219-N1 by HPLC–reverse phase. The mean purity for the 3 identical runs was 98.5% with a %CV of 1.2%.

Figure 3

Integrity assessment for purified RBD by SDS-PAGE under reducing and nonreducing conditions followed by (a) Coomassie Blue staining: 2-6 μg of purified RBDs were loaded, (b) Silver staining: 0.5-5 μg of purified RBDs were loaded, or (c) Western blot: transferred to polyvinylidene difluoride membrane and probed with an RBD-specific antibody (33G4); 2-6 μg of purified RBDs were loaded.

Figure 4

(a) Circular dichroism results for RBD219-N1 at 25°C, (b) CD profile for RBD219-N1 at different temperatures, (c) CD denaturation profile and the first CD derivative of RBD219-N1 at 220 nm, and (d) CD denaturation profile and the first CD derivative of RBD219-N1 at 230 nm.

Figure 5

(a) Thermal shift assay results and (b) the derivatives of the intensity for purified RBD219-N1 lots. DI water was used as negative control.