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\begin{document}

\title{Production of high-quality SARS-CoV-2 antigens: impact of bioprocess and
storage on glycosylation, biophysical attributes, and ELISA serologic
tests performance}



\author[1]{Rute Castro}%
\author[1]{Lígia Nobre}%
\author[1]{Rute Eleutério}%
\author[1]{Mónica Thomaz}%
\author[1]{António Pires}%
\author[1]{Sandra Monteiro}%
\author[1]{Sónia Mendes}%
\author[2]{Ricardo  Gomes}%
\author[1]{João Clemente}%
\author[1]{Marcos Sousa}%
\author[1]{Filipe Pinto}%
\author[1]{Ana Silva}%
\author[1]{Micael Freitas}%
\author[1]{Ana Lemos}%
\author[3]{Onome Akpogheneta}%
\author[3]{Lindsay  Kosack}%
\author[3]{Marie-Louise  Bergman}%
\author[3]{Nadia Duarte}%
\author[3]{Paula Mastoso}%
\author[4]{Julia Costa}%
\author[1]{Tiago Bandeiras}%
\author[5]{Patricia Gomes-Alves}%
\author[3]{Carlos Gonçalves}%
\author[3]{Jocelyne Demengeot}%
\author[1]{Paula Alves}%
\affil[1]{iBET, Instituto de Tecnologia Experimental e Tecnológica}%
\affil[2]{Instituto de Biologia Experimental e Tecnologica}%
\affil[3]{Instituto Gulbenkian de Ciencia}%
\affil[4]{Instituto de Tecnologia Quiimica e Biologica}%
\affil[5]{iBET/ ITQB-NOVA}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\begin{abstract}
SARS-CoV-2 is an RNA coronavirus that causes severe acute pneumonia,
also known as COVID 19 disease. The World Health Organization declared
the COVID-19 outbreak in January 2020 and a pandemic 2 months later.
Serological assays are valuable tools to study virus spread among the
population and, importantly, to identify individuals that were already
infected and would be potentially immune to a virus re-infection.
SARS-CoV-2 Spike protein and its Receptor Binding Domain (RBD) are the
antigens with higher potential to develop SARS-CoV-2 serological assays.
Moreover, structural studies of these antigens are key to understand the
molecular basis for Spike interaction with angiotensin converting enzyme
2 receptor, hopefully enabling the discovery and development of COVID-19
therapeutics. Thus, it is urgent that significant amounts of this
protein became available at the highest quality. In this work we
evaluated the impact of different and scalable bioprocessing approaches
on Spike and RBD production yields and, more importantly, in these
antigens' quality attributes. Using negative and positive sera collected
from human donors, we show an excellent performance of the produced
antigens, assessed in serologic ELISA tests, as denoted by the high
specificity and sensitivity of the test. We have shown that, despite of
the human cell host and the cell culture strategy used, for production
scales ranging from 1 L to up to 30 L, final yields of approx. 2 mg and
90 mg per liter of purified bulk for Spike and RBD, respectively, could
be obtained. To the best of our knowledge these are the highest yields
for RBD production reported to date. An in-depth characterization of
SARS CoV-2 Spike and RBD proteins was also performed, namely the
antigens oligomeric state, glycosylation profiles and thermal stability
during storage. The correlation of these quality attributes with ELISA
performance show equivalent reactivity to SARS CoV 2 positive serum, for
all Spike and RBD produced, and for all the storage conditions tested.
Overall, we provide herein straightforward protocols to produce
high-quality SARS CoV-2 Spike and RBD antigens, that can be easily
adapted to both academic and industrial settings; and integrate, for the
first time, studies on the impact of bioprocess with an in-deep
characterization of these proteins, correlating antigens glycosylation
and biophysical attributes to performance of COVID-19 serologic tests.
We strongly believe that our work will contribute to advance the current
and recent knowledge on SARS-CoV-2 proteins and support the scientific
society that is persistently searching for solutions for COVID-19
pandemics.%
\end{abstract}%



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