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\begin{document}

\title{Genome-scale modeling of CHO cells unravel the critical role of
asparagine in cell culture feed media}



\author[1]{Kuin Tian Pang}%
\author[1]{Yi Fan Hong}%
\author[2]{Fumi Shozui}%
\author[2]{Shunpei Furomitsu}%
\author[1]{Matthew Myint}%
\author[3]{Ying Swan Ho}%
\author[4]{Yaron R.  Silberberg}%
\author[1]{Ian Walsh}%
\author[5]{Meiyappan  Lakshmanan}%
\affil[1]{Bioprocessing Technology Institute}%
\affil[2]{Ajinomoto Co Inc}%
\affil[3]{Bioprocessing Research Industry Club}%
\affil[4]{Ajinomoto CELLiST Korea, Co., Inc,}%
\affil[5]{Indian Institute of Technology Madras}%


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  \date{May 24, 2024}


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\begin{abstract}
Amino acids, including asparagine, aspartate, glutamine, and glutamate,
play important roles in the purine and pyrimidine biosynthesis as well
as serve as anaplerotic sources fueling the tricarboxylic acid (TCA)
cycle for mitochondrial energy generation in mammalian cells. Despite
extensive studies on glutamine and glutamate in CHO cell cultures, the
roles of asparagine and aspartate, especially in feed media, remain
underexplored. In this study, we utilized the CHO genome scale model to
first deeply characterize the intracellular metabolic states of CHO
cells cultured in different combinations of basal and feed media to
understand the traits of asparagine/aspartate-dependent and
glutamate-dependent feeds. Subsequently, we identified the critical role
of asparagine and aspartate in the feed media as anaplerotic sources and
conduct in silico simulations to ascertain their optimal ratios to
improve cell culture performance. Finally, based on the model
simulations, we reformulated the feed media by tailoring the
concentrations of asparagine and aspartate. Our experimental data reveal
a CHO cell preference for asparagine compared with aspartate, and thus
maintaining an optimal ratio of these amino acids is a key factor for
achieving optimal CHO cell culture performance in biopharmaceutical
production.%
\end{abstract}%



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