2 × 0. 5 µl of the sample / matrix mixture were then spotted onto a clean matrix ‐ assisted laser desorption ionization ( maldi ) plate and dried under vacuum. the spotted plate was then loaded into a 4800 maldi ‐ tof / tof mass spectrometer ( applied biosystems, usa ) for ms and ms / ms glyco mic analyses. maldi ‐ ms data were collected in the positive ion mode, with a mass range of m / z 500 – 9000. four spectra ( 1000 shots each ) were collected for each sample / spot with a laser intensity of 4000 and 20 kv accelerating voltage. ms / ms analyses were performed on the same instrument, however, using the positive reflectron mode, with argon gas in the collision cell ( at 10mbar pressure ) to induce collisionally activated decomposition. for these experiments, the potential difference between the source acceleration voltage and the collision cell was set to 1 kv. all structural assignments of n ‐ glycans were based on molecular ion composition, previous knowledge of biosynthetic pathways, and ms / ms fragmentation patterns. data explorer ( applied biosystems, usa ) and glyco workbench were used for the analysis of maldi ‐ ms and maldi ‐ ms / ms spectra ( ceroni et al. 2008 ). quantitative information of n ‐ glycans was derived from ion counts which do not consider ionization efficiencies and should therefore not be considered absolute values. the relative abundances of galactose and neuac sialic acid in each sample were calculated by summing the relative intensities of galactose ‐, or sialic acid ‐ containing n ‐ glycans and dividing by the sum of relative intensities for all assigned n ‐ glycans and finally expressed as a percentage ( the number of gal or neuac residues in each structure were not considered in this analysis ) ( see figures 3 and 8 ). the same method was used to analyze the relative abundance of core ‐ fucosylation, and oligomannose / complex n ‐ glycans. this data omit monoisotopic peaks corresponding to putative very low abundance afucosylated structures that might be present in isotopic clusters ( see figures s5 and s6 ). samples for n ‐ glyco mic analyses were prepared from cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cell cultures grown in suspension that were pelleted, washed, and sonicate d. glyco proteins in the cell lysates were then reduced and carboxymethylated, and tryptically digested. the n ‐ glycans were subsequently released from tryptic glyco peptides by pngase f digestion, purified, and permethylated to increase the sensitivity of the ms analysis. endo ‐ β ‐ galactosidase enzymatic digests were also performed on purified n ‐ glycans prior to permethylation to enhance the structural characterization of cellular n ‐ glycans from cho cells. this enzyme cleaves the internal β1, 4 ‐ gal linkage of antennal lacnac ' s, and the resulting ms profiles offer additional insights into complex n ‐ glycan branching and terminal elaboration patterns. finally, the n ‐ glycans from purified igg1 ‐ fc constructs produced in cho ‐ s cells were also analyzed using ms and compared to previously published data for the same constructs produced in cho ‐ k1 cells ( blundell et al. 2019, 2020 ). we used maldi ‐ ms to generate singly charged sodiated molecular ions ( [ m + na ] ). the ms spectra generated were then structurally annotate d by mass fingerprinting and used to perform a semi ‐ quantitative analysis of relative intensities of different n ‐ glycan structures. our analysis was primarily focused on comparing the relative abundances of oligomannose n ‐ glycans and complex n ‐ glycans with variable levels of fucosylation, galactosylation, and sialylation and different branching patterns. furthermore, certain molecular ions detected in the ms spectra were analysed by ms / ms. through collisionally activated dissociation, ms / ms generates sequence ‐ informative fragment ions, which help to identify or confirm antennal structures, branching patterns, and linkage positions. the low m / z range of the n ‐ glycan spectra of cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cells shows prominent signals corresponding oligomannose structures man5glcnac2 to man9glcnac2 ( m / z 1579, 1783, 1988, 2192, and 2396 ), with a range of 70 % – 78 % of summed monoisotopic peak intensities ( see figures 1 and 3, table 2 ). additionally, the n ‐ glyco mes of all the cell lines encompass a diverse array of complex n ‐ glycans, both with and without core fucose, featuring bi ‐, tri ‐, or tetra ‐ antennary branching ( see figures 2 and 3 ). these structures also exhibit variable ‐ length antennal lacnac extensions, with up to four terminal sialic acids residues on tetra ‐ antennary n ‐ glycans. although the majority of sialic acids were neuac, some minor peaks suggest the presence of n ‐ glyco lylneuraminic acid ( neugc ) as well ( e. g., m / z 2996, 3084 — see figures s1 – s4 for full annotation ). while expression of the enzyme cmp ‐ n ‐ acetylneuraminic acid hydroxylase, responsible for neugc production, has previously been shown to be virtually absent in cho ‐ k1 cells, it is thought that some residual activity may be responsible for the presence of neugc ‐ sialylated structures ( north et al. 2010; xu et al. 2011 ). fortunately, neugc levels in cho cells and the biotherapeutics produced in these hosts are believed to be negligible, thus avoiding the potentially harmful immunogenic effects of this epitope in humans ( ghaderi et al. 2010, 2012; tangvoranuntakul et al. 2003 ). maldi ‐ tof ‐ tof ms / ms analyses were used to verify the presence of variably branched structures and polylactosamine extended antennae ( see figures s5 and s6 ). additionally, ms / ms compositional analyses confirmed that only core ‐ fucose was present in cho cells ( north et al. 2010 ). more broadly, the ms data obtained in this study reflect a similar range of n ‐ glycan structures to those previously identified in pro5 cho cells ( north et al. 2010 ). however, upon closer inspection, the data obtained in this study reveal distinc t patterns in the size and antennal branching of n ‐ glycans. for instance, the sialic acid content of cho ‐ k1 and cho ‐ s cells is significantly higher than in cho pro5 cells. this can be readily observed from the fact that sialylated structures have the highest relative intensities among the complex n ‐ glycans in cho ‐ k1 and cho ‐ s cells, whereas in cho pro5 cells, asialylated structures were more abundant than the sialylated counterparts ( north et al. 2010 ). these differences can most likely be explained by the impact of genetic divergence and antibod y production / secretion on the glyco sylation machinery of cho ‐ k1 and cho ‐ s cells. the inherent genetic variations between cho cell lines fundamentally influence their posttranslational modification capabilities, as each cell line possesses distinc t genetic backgrounds that affect protein processing pathways ( reinhart et al. 2018 ). multiomics analyses have demonstrated that physiological variations among cho cell lines cultured under identical conditions stem from underlying genetic deficits that impact cellular processes, including glyco sylation machinery ( lakshmanan et al. 2019 ). these genetic differences result in varied expression patterns of glyco sylation enzymes, ultimately affecting the n ‐ glycan profiles produced by different cho cell lines ( lewis et al. 2013 ). additionally, it has been suggested that the relative abundance of glyco sylation machinery to cellular secretory capacity may be a critical determinant of glyco sylation efficiency, linking the genetic expression profile to higher ‐ level cellular dynamics ( jimenez del val et al. 2016 ).