An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein.

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.