Chinese Hamster Ovary (CHO) cells are the leading mammalian cell platform for manufacturing therapeutic proteins due to their ability for producing human-like products. However, relatively high production costs compared to other non- eukaryotic systems (bacteria, yeast, etc.), genetic instability and inefficient metabolism limit productivity. Strategies have been developed to improve yields in a cost effective manner, such as media optimization, improved feeding regimes, genetic engineering, among others, but further improvement is desirable. This thesis focused on characterizing the central carbon metabolism of different recombinant CHO cells to identify novel molecular mechanisms by increasing the metabolic load overexpressing human erythropoietin (EPO). Metabolomics data obtained through gas chromatography mass spectrometry (GS-MS) of a tetracycline (tet) inducible CHO T-RExTM cell line capable of expressing 0.09 pg/ul/cell EPO, revealed that 0.1% v/v tet addition does not affect consumption of carbon sources such as glucose, and pyruvate, but concentration of waste products, such as glycerol, threitol, alanine and phenyllactic acid increases towards the end of each batch culture. Tricarboxylic acid cycle (TCA) intermediates, such as citrate, isocitrate and fumarate also increased upon EPO expression. Additionally, two DG44 cell lines containing inducible (pcDNA4/TO) or constitutive (pCDNA3.1) vectors (DG44-I and DG44-C cells respectively) with EPO and DHFR genes were generated. In order to trigger gene amplification of both genes, DG44-I and DG44-C cells were incubated and sub cultured in the presence of 250 nM MTX for 35 days, leading to EPO titers of 0.123 and 0.15 pg/ul/cell respectively without affecting maximum viable cell density (VCD). Metabolomics data obtained through GC-MS showed that 250 nM MTX and 0.1% (v/v) tet do not affect consumption of carbon and energy sources such as glucose, serine, pyruvate and many others, but accumulation of glycine and glycerol are significantly higher in batch cultures. TCA intermediates such as citrate and isocitrate also accumulate more in the same conditions. Glycerol, citrate and isotritate significantly increased in all the cell lines assessed upon EPO expression. As a result, reducing waste product production through down-regulating GDPH, an enzyme directly related with glycerol production, as well as overexpressing TCA enzymes such as isocitrate dehydrogenase and aconitase, could potentially increase carbon flux, reduce waste products production and potentially increase productivity of recombinant proteins.