Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain\u2013based vaccine candidate, RBD219-N1C1

Jungsoon Lee,Bin Zhan,Maria Elena Bottazzi,Joanne Altieri Rivera,Rakesh Adhikari,Ulrich Strych,Peter J Hotez,Rakhi Kundu,Zhuyun Liu,Junfei Wei,Wen-Hsiang Chen,Portia M Gillespie

doi:10.1007/s00253-021-11281-3

Abstract

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.Key points• Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined.• Three purification protocols for a COVID-19 vaccine antigen were compared.• Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown.Graphical abstract

Highlights

After the first report of coronavirus disease 2019 (COVID-19) in December 2019 (Li et al 2020; Lu et al 2020), the number of cases is at 124 million with over 2.3 million deaths worldwide (JHU 2021)
When basalsalt medium (BSM) and low-salt medium (LSM) were compared for the production of the RBD219-N1C1 protein, no differences were observed in the growth profiles and the final biomass
We developed a process suitable for producing a recombinant protein COVID-19 vaccine antigen for clinical testing and transition to industrial manufacture

Summary

Introduction

After the first report of coronavirus disease 2019 (COVID-19) in December 2019 (Li et al 2020; Lu et al 2020), the number of cases is at 124 million with over 2.3 million deaths worldwide (JHU 2021). Moderna, both produced in record time, but posing the challenges that are relatively expensive to manufacture and difficult to scale and require transportation and storage at ultra-low temperatures. The viral vector–based vaccines developed by Oxford-AstraZeneca, Johnson & Johnson (J&J), Gamaleya, and CanSino have been approved for use in a variety of countries and bring the added advantage that do not have these ultra-low cold-chain requirements providing easier delivery and, in the case of the J&J vaccine, the advantage of being used as a single dose. Several whole inactivated viral vaccines produced by the Chinese manufacturers Sinopharm and Sinovac and by Bharat Biotech, an India-based manufacturer, have added to the list of approved vaccines (Craven 2020; Eccleston-Turner and Upton 2021). Data from Duke’s Global Health Innovation Center and others clearly highlight the continued procurement and manufacturing challenges, leading to enormous inequity in the access for COVID-19 vaccines in these regions of the world (Duke Global Health Innovation Center 2021; Ritchie et al 2021)

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