additionally, the cho ‐ k1mab cells also show a statistically significant increase in sialylation compared to cho ‐ smab cells ( 21 % vs. 14 %, * p < 0. 05 ). a good example of this increased sialylation can be observed when comparing the ratios of the m / z 2605 and 2966 signals, which correspond to mono ‐, and di ‐ sialylated, core ‐ fucosylated biantennary complex n ‐ glycans, respectively ( see figure 2 ). indeed, in cho ‐ k1 and cho ‐ k1mab cells, the peak for the disialylated n ‐ glycan at m / z 2966 is roughly 3× larger than the m / z 2605 peak, whereas in cho ‐ s and cho ‐ smab cells, the m / z 2966 signal is actually the smaller of the two. the contrast between the antibod y ‐ producing cell lines is less pronounced than the differences between their parental cho ‐ k1 and cho ‐ s cell lines. this observation is linked to another pattern: antibod y production has inverse effects on the levels of complex n ‐ glycans in these cell lines, although as mentioned previously, only differences in sialylation levels were found to be statistically significant. in fact, core ‐ fucosylation, galactosylation, and sialylation levels are lower in cho ‐ k1mab cells than cho ‐ k1 cells, whereas the opposite applies to cho ‐ smab and cho ‐ s cells. it is difficult to explain exactly why these opposing trends are observed and further investigation is warranted. one hypothesis is that if antibod y production does influence n ‐ glyco sylation in cho cells, it is likely to be linked to the specific mab productivity of the cell, which was not investigated in this study. we envisage that specific mab productivity is likely to affect glyco sylation enzyme saturation and glyco protein residence time in various golgi cisternae along the secretory pathway, which could affect n ‐ glycan processing in host cell glyco proteins ( jimenez del val et al. 2016 ). additionally, the consumption of cellular resources for mab production might affect the expression of glyco syltransferases, glyco sidases, sugar transporters, and upstream metabolic pathways linked to glyco sylation. for example, when the specific productivity of various biopharmaceuticals was increased in cho cells via temperature reduction or sodium butyrate supplementation, variable effects on glyco sylation were reported ( ahn et al. 2008; fox et al. 2004; hendrick et al. 2001; kantardjieff et al. 2010; spearman et al. 2005; sung et al. 2004; trummer et al. 2006 ). these results were generally considered a consequence of pleiotropic effects linked to cell cycle arrest. conversely, some articles suggest that increased specific productivity is linked with reduced glycan processing ( fan et al. 2015; santell et al. 1999; trummer et al. 2006 ). one group in particular concluded that this was likely to be a result of reduced availability of glyco sylation machinery relative to the secretory capacity of the cell ( fan et al. 2015; jimenez del val et al. 2016 ). the underlying mechanistic details of how mab production might influence cellular n ‐ glyco sylation and biopharmaceutical glyco sylation are poorly understood. further studies will be necessary for understanding whether effects are cell line ‐ specific and how glyco sylation machinery availability, golgi apparatus size, and retention time might affect glyco sylation capabilities. an additional level of structural characterization of the n ‐ glyco me in cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cells was carried out using endo ‐ β ‐ galactosidase digests to better determine differences in nonreducing end terminal motifs, and branching patterns. endo ‐ β ‐ galactosidase hydrolyzes internal β1 ‐ 4 linkages in linear lacnac repeats ( [ glcnac β1 ‐ 3galβ1 ‐ 4 ] n ) and the postdigestion maldi ‐ tof ms spectra are presented in figure 4. the enzyme treatment generates signals corresponding to the structures of trimmed antennal motifs ( m / z 518 — glcnacgal, m / z 722 — galglcnacgal, and m / z 1083 — neuacgalglcnacgal ), which provide an additional data set for comparing the relative abundances of galactosylated and sialylated polylacnac antennae. additionally, endo ‐ β ‐ galactosidase digests also produce bi ‐, tri ‐, and tetra ‐ antennary trimmed core n ‐ glycans with signals at m / z 1836, 2081, 2285, 2326, and 2530 that can help the comparison of branching levels of polylacnac containing glycans across the cell lines. the results show that these cho cell lines present polylactosamine extensions on a mixture of bi ‐, tri ‐, and tetra ‐ antennary n ‐ glycans, similarly to previous findings ( north et al. 2010 ). maldi ‐ tof ms profiles of permethylated n ‐ linked glycans derived from cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cells after endo ‐ β ‐ galactosidase digestion of released n ‐ glycans. all molecular ions represent the singly charged and sodiated form [ m + na ]. data in the m / z 500 – 1500 range were obtained from the 35 % mecn fraction from a c18 sep ‐ pak. data in the m / z 1600 – 2600 range were obtained from the 50 % mecn fraction. below the maldi ‐ tof ms spectra is a diag ram showing an example of the expected n ‐ glycan and released antennal structures resulting from endo ‐ β ‐ galactosidase digestion of a complex n ‐ glycan. the semi ‐ quantitative analysis of n ‐ glycans from digested samples revealed that lacnac extensions on tri ‐ antennary structures were significantly more abundant in cho ‐ s and cho ‐ smab cells ( e. g., m / z 2081 ) compared to cho ‐ k1 and cho ‐ k1mab cells, whilst polylactosamine repeats were considerably more abundant in bi ‐ and tetra ‐ antennary n ‐ glycans ( e. g., m / z 1836, 2326 ) in the latter pair ( see figure 5 ). this contrasts with previous data from pro5 cho cells, where polylactosamine extensions were found primarily on biantennary n ‐ glycans ( north et al. 2010 ). interestingly, both mab ‐ producing cell lines show a greater abundance of tetra ‐ antennary n ‐ glycans and a lower abundance of tri ‐ antennary structures than in parental cell lines. more specifically, cho ‐ k1mab cells show a greater abundance of the tetra ‐ antennary n ‐ glycan signal at m / z 2530 ( * p < 0. 05 ) and a lower abundance of the tri ‐ antennary n ‐ glycan at m / z 2081 ( * * p < 0. 01 ) compared with cho ‐ k1 cells. similarly, the levels of tetra ‐ antennary structures were higher in cho ‐ smab cells than the parental cho ‐ s cells ( m / z 2326, * * * p < 0. 001 ), whereas tri ‐ antennary n ‐ glycans were less abundant ( m / z 2285, * * p < 0. 01 ). overall, these findings indicate a correlation between antibod y production and increased branching in n ‐ glycans in both cho ‐ k1 and cho ‐ s cells. graphical detail of terminal motif distribution and antennarity of n ‐ glycans, after endo ‐ β ‐ galactosidase digest, from cho ‐ k1 and cho ‐ s cell lysates compared with antibod y ‐ producing cho ‐ k1mab and cho ‐ smab cells. the left ‐ hand graph represents n ‐ glycan profiles obtained from 35 % mecn fractions from a c18 sep ‐ pak, while the right ‐ hand graph refers to the 50 % mecn profiles. terminal structures with m / z 518, 722, and 1083 do not elute well in the 50 % mecn fraction, which is why these data are not shown. the structures assigned to the various molecular ions are shown. the relative intensities are the mean of three biological replicates and error bars represent ± 1 sd. the results of two ‐ tailed unpaired t ‐ tests are shown above the columns ( n = 3 / group, nonsignificant ( ns ) = p > 0. 05, * p < 0. 05, * * p < 0. 01, * * * p < 0. 001, * * * * p < 0. 0001 ). in line with data from the undigested samples, the cleaved sialylated terminal motif at m / z 1083 is more abundant ( ~ 2×, * * p < 0. 01 ) in cho ‐ k1 cells than in cho ‐ s cells ( see figure 5 ), confirming the greater sialylation levels in cho ‐ k1 cells.