ABSTRACT: Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets for improved RP production and quality. A special emphasis has been placed on proteomic characterization of subcellular compartments over cellular homogenates, due to some advantages that fractionation techniques offer over the classical approach, which include obtaining datasets that are easier to process and understand. Subcellular proteomics allows monitoring of proteins that shuttle between different compartments, quantification of lower abundance proteins, increase in number of identified proteins, elucidation of function and regulation of proteins, and unraveling of organelle dynamics. The use of subcellular fractionation for proteomic characterization of organelles from CHO cells has been limited to the isolation of one or few organelles in an adherent phenotype, and only few fractionation protocols have been reported for these cells in their suspension format. The high cross-contamination of some isolated fractions due to insufficient fractionation steps, prevents adopting some of these protocols for applications such as proteomics. Thus, since this approach has not been addressed before for the proteomic characterization of CHO cell organelles, subcellular proteomics was harnessed in the present study to provide identification and quantification of proteins from subcellular compartments obtained by differential and isopycnic centrifugation of this cell line. Although several cellular processes and organelles play an important role during expression and secretion of RP, the classical secretion pathway has been recognized as a bottleneck for post-translational modifications, transport, modification and secretion of proteins in mammalian cells. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the specific productivity of HEK293 and CHO cells producing different RP. Thus, in line with the paramount importance of organelles from classical secretion pathway for RP production, a novel discontinuous sucrose gradient for improved separation and enrichment of microsomes has been designed in our laboratory, and incorporated to the proteomic characterization of CHO cell organelles. CRL-12444, a DP-12 derived cell line producing a humanized monoclonal antibody against human IL-8, was used as a model of a recombinant CHO cell line during this study. Our goal is that the processing of raw proteomics data, the classification and mapping of identified proteins will allow to construct proteomic maps for most subcellular compartments, describing the molecular functions, biological processes and pathways from enriched fractions. The identification of proteins in each enriched compartment will improve the knowledge about their protein components, function and interaction, especially from those poorly represented like the proteins in the microsomes, allowing a subsequently expansion of the engineering strategies of CHO cells to increase the titer and quality of RPs.
Saumel Perez Rodriguez 
