The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has revealed the need to develop technologies that are capable of rapidly responding to emerging biological threats. A key component of this response includes the ability to produce vaccines quickly, at an affordable cost, and at scale for equitable global access. In this regard, antigen subunit vaccines against SARS-CoV-2 are advantageous over mRNA vaccines because proteins are often easier to produce at scale and require a less robust cold chain to maintain efficacy, thus, facilitating production and distribution in middle and low-income countries.

Major antigen subunit vaccine production platforms approved for use as vaccines include Pichia pastoris, CHO cells, HEK-293T cells, plant, and Sf9 insect cells which all suffer in varying degrees to labor-intensive and expensive manufacturing processes along with relatively low yields of antigen production. As an alternative, a S1-RBD antigen was produced in a bioreactor using a proprietary genetically modified C1 clone of Thermothelomyces heterothallica and purified using C-Tag affinity chromatography.¹ The purified S1-RBD antigen was adjuvanted with Alhydrogel^®‘85’ to produce vaccine candidate DYAI-100A85 that is currently in Phase I trials to demonstrate safety in humans.²

In the present work, the same C1 clone is used to produce S1-RBD in a 5L bioreactor and purified using an anti-RBD affinity chromatography. This purified S1-RBD was then characterized using biolayer interferometry to determine association and dissociation rate constants of RBD and ACE-2, thermal shift to determine thermal stability, circular dichroism to determine secondary protein structure, and MALDI-TOF to determine peptide mass. Additionally, we have mapped the glycans of C1 produced S1-RBD and demonstrated the ability to modify this glycan structure, if necessary. A molecular dynamics model of this C1 S1-RBD has been developed and is being used to characterize the structure and the influence of glycosylation.

Shruthi Garimella

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