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\begin{document}

\title{Combined multivariate statistical and flux balance analyses uncover
media bottlenecks to the growth and productivity of CHO cell cultures}



\author[1]{Hock Chuan  Yeo}%
\author[2]{Seo-Young Park}%
\author[1]{Tessa Tan}%
\author[1]{Say Kong Ng}%
\author[1]{Meiyappan  Lakshmanan}%
\author[2]{Dong-Yup Lee}%
\affil[1]{Bioprocessing Technology Institute}%
\affil[2]{Sungkyunkwan University College of Natural Science}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\begin{abstract}
Chinese hamster ovary (CHO) cells are widely used for producing
recombinant proteins. To enhance their growth, productivity, and product
quality, practically media reformulation has been one of key focuses
with several technical challenges which are due to the myriad of
intricate molecular and regulatory mechanisms underlying the media
effects on culture behaviours; it is highly required to systematically
characterize metabolic bottlenecks of cell cultures in various media
conditions. To do so, we combined multivariate statistical analysis with
flux balance analysis of a genome-scale metabolic model of CHO cells
based on the culture profiles of CHO-DG44 under one commercial medium
and two in-house media. At the outset, we used partial least square
regression to identify metabolite exchanges that are correlated to
specific growth and productivity. By using a commercial medium as
reference, we found sub-optimal level of four nutrients and two
metabolic wastes that plausibly hinder cell growth and productivity with
in-house media. Subsequently, we elucidated that the recycling of
lactate and ammonia wastes to be affected by both glutamine and
asparagine metabolisms mechanistically, and further modulated by
hitherto unsuspected folate and choline supplements. In summary, the
current work successfully demonstrated how multivariate statistical
analysis can be synergistically combined with in silico analysis of
metabolic models to uncover the mechanistic elements underlying the
differing performance of various media. Our approach for the systematic
identification of promising nutrient targets thus paves the way for cell
culture medium reformulation to enhance cellular growth and recombinant
protein production.%
\end{abstract}%



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