lllumina data and comparison to proteomic databaseexpression levels of proteins detected by the powerblot assay were compared to our previous published database of multiple hesc lines examined using the illumina bead array platform ( liu et al., 2006 ). averaged transcript expression signals from the bg01, bg02 and bg03 cell lines were converted to a + / - format, based on the following criteria: a mean transcript detection level of > 5, 000 was designated as + + + +; 1, 000 – 5, 000 as + + +; 100 – 1, 000 as + +; 30 – 100 as +; and signals < 30 was represented as -. in parallel, the protein expression levels were converted to a + / - format based on these criteria: i. u. > 10, 000 as + + + +; 5, 000 – 10, 000 as + + +; 1, 000 – 5, 000 as + +; 100 – 1, 000 as +. in addition, genes were categorized into the same functional / signaling pathways as per the western blot database. while chinese hamster ovary ( cho ) cells continue to be the workhorse of recombinant therapeutic protein production, decades of genetic divergence in the industrially relevant cho ‐ k1 and cho ‐ s cell lines, are likely to have resulted in differing glyco sylation capabilities. glyco sylation can influence the efficacy, serum half ‐ life, and safety of biologics, and as a critical quality attribute of glyco protein biopharmaceuticals, it is essential to better understand how major cho cell manufacturing platforms diverge. we used matrix ‐ assisted laser desorption ionization ‐ time of flight mass spectrometry to perform n ‐ glyco mic analyses comparing cho ‐ k1 cells, cho ‐ s cells and antibod y ‐ producing daughter cell lines. the results reveal that genetic divergence in these industrially relevant cell lines, as well as the burden of antibod y production, lead to significant differences in antennal branching and terminal elaboration in the cellular n ‐ glyco me. more specifically, cho ‐ k1 cells produce larger and more complex n ‐ glycans with higher levels of sialylation than cho ‐ s cells, and antibod y production was associated with increased antennal branching. additionally, these findings were also reflected in the n ‐ glyco mic profiles of igg1 ‐ fc constructs produced in either cho ‐ k1 and cho ‐ s cells. chinese hamster ovary ( cho ) cells are currently the most important manufacturing platform for the multibillion usd biopharmaceutical industry. from 2018 to 2022, nearly 70 % of approved biopharmaceuticals, and 85 % of novel macromolecules ( i. e., excluding biosimilars ), were produced in cho cells ( g. walsh and e. walsh 2022 ). an even larger proportion of monoclonal antibod ies ( mabs ), 89 %, were produced in these hosts ( g. walsh and e. walsh 2022 ). in the decades since the first biopharmaceuticals were clinically approved, cho cells came to dominate the manufacturing landscape partly because of human ‐ compatible glyco sylation profiles and high production titers ( 10 – 15 g∙l ) ( puetz and wurm 2019 ). despite the advantages of such a platform, glyco sylation is not a template ‐ driven process and remains a heterogenous affair even in cho cells. enzyme competition for substrates in the cellular glyco sylation reaction networks generates a mixture of glycan structures on glyco protein therapeutics. n ‐ and o ‐ glyco sylation both directly and indirectly modulate biopharmaceutical efficacy, serum half ‐ life, and immunogenic responses, which is why increased control over cho cell glyco sylation and in vivo glyco engineering are useful strategies that require a deep understanding of cho cell glyco sylation capabilities. while glyco sylation is not present in all recombinant therapeutic proteins and can be engineered out when functionally redundant, it remains a critical quality attribute of glyco protein therapeutics under the fda ' s “ quality by design ” paradigm. in fact, reproducible glyco sylation profiles are essential for the development of biosimilars that ac curate ly mimic originator products. furthermore, glyco engineering can serve as a foundation for diverse strategies aimed at enhancing the quality of biopharmaceuticals in the development of biobetters. a previous mass spectrometry ( ms ) glyco mic analysis of n ‐ and o ‐ glycans from cho ‐ pro5 cells and a panel of glyco sylation mutants forms the basis of our current understanding of the cho cell glyco sylation machinery ( north et al. 2010 ). the data reported only simple mono ‐ or disialylated core ‐ 1 o ‐ glycan structures ( + / −neuaca2, 3galβ1, 3 ‐ ( + / −neuaca2, 6 ) galnacα ‐ ser / thr ) were produced by cho cells, however, the n ‐ glyco mic profiles revealed spectra with high mannose ( man ) n ‐ glycans together with a wide range of complex n ‐ glycans. in cho cells, the complex n ‐ glyco me includes a mixture of bi ‐, tri ‐, and tetra ‐ antennary n ‐ glycans that can form large structures with highly extended antennae including up to 26 lactosamine ( lacnac; gal ‐ glcnac ) units. the antennae can also be capped by n ‐ acetylneuraminic acid ( neuac sialic acid ) and fucose ( fuc ) was found to be present only in the chitobiose core. while these experiments generated a wealth of glyco mic data for cho cells, the pro5 cells in this study are not the dominant cell line used in industry for the manufacturing of biopharmaceutical products. in fact, cho ‐ k1 and cho ‐ s cells are some of the most commonly used cell lines for industrial bioprocesses in the pharmaceutical industry ( f. wurm and m. wurm 2017 ). it is not possible to exactly determine the precise pedigree of cho cell lines in use today, however, a review by wurm and wurm traces the origins of the cho ‐ k1 cell line to the late 1960s, whereas the cho ‐ s cell line emerged in the 1970s ( f. wurm and m. wurm 2017 ). the initially immortalized cho cells, and all the cho cell lines that have since emerged, were released from the constraints imposed by diploidy control, and subsequently evolved with higher rates of dna mutations and changes in gene expression profiles ( f. wurm and m. wurm 2017 ). inherent genetic instability in cho cells and a lack of “ true ” clonality is not necessarily an issue for the reliable production of high ‐ quality biopharmaceuticals. however, it is likely that over decades of culturing — often in a variety of conditions that alter selective pressures — and many cell division events, the genetic divergence between, and indeed, within cho “ cell lines ” could be of significance. while clonal selection is a standard process in industry that includes glyco sylation as a selection criterion, genetic divergence may increase with each cell division event, potentially leading to heterogenous glyco sylation capabilities and loss of “ clonality. ” we posited that such genetic divergence might be reflected in the expression levels of glyco sylation genes and consequently affect the n ‐ glyco sylation profiles of different cho cell lines and the recombinant glyco protein therapeutics produced in these cells. a genomic comparison of the cho ‐ k1, cho ‐ s, and cho ‐ dg44 cell lineages previously highlighted such genetic differences and suggested that further biochemical characterization of glyco mic phenotypes was necessary ( lewis et al. 2013 ). herein, we present mass spectrometric n ‐ glyco mic data that highlight differences in cellular n ‐ glyco sylation in two industrially important cho cell lineages: cho ‐ k1 and cho ‐ s. our focus on n ‐ glycan profiles stems from the fact that most glyco sylation in recombinant biopharmaceuticals, such as igg mabs, is n ‐ linked. additionally, cho cell o ‐ glyco sylation is limited to simple core ‐ 1 structures with or without sialic acid capping and is less informative about the overall glyco sylation machinery of cho cells than n ‐ glycans. furthermore, we analyzed the n ‐ glyco me of igg mab ‐ producing cells derived from cho ‐ k1 and cho ‐ s cells to determine whether the production and secretion of mabs can itself influence the glyco sylation machinery of cho cells.