a lower abundance of the terminally galactosylated motif at m / z 722 in cho ‐ k1 cells relative to cho ‐ s cells ( * * * p < 0. 001 ) is further evidence of greater sialyltransferase activity in the k1 lineage. the same difference is observed between the antibod y ‐ producing cell lines, although it is less pronounced ( * p < 0. 05 ). sialylation levels based on the m / z 1083 terminal motif were also found to be lower in cho ‐ k1mab cells relative to the progenitor cho ‐ k1 cells ( * * p < 0. 01 ), which reflects findings from the undigested samples. the lower abundance of uncapped terminal motifs at m / z 518 and 722 in cho ‐ k1 cells compared with the antibod y ‐ producing daughter cell line ( * p < 0. 05 and * * p < 0. 01, respectively ) lends support to the hypothesis that antibod y production is correlated with decreased sialylation in cho ‐ k1 cells. in addition to comparing the cellular n ‐ glyco sylation capabilities of cho ‐ k1 and cho ‐ s cells, we used maldi ‐ tof ms to compare n ‐ glyco mic profiles from a series of igg1 ‐ fc constructs produced in cho ‐ s cells to previously obtained data for the same constructs in cho ‐ k1 cells ( blundell et al. 2019, 2020 ). protein backbone mutations are an established glyco engineering strategy, which can be used to add novel glyco sylation sites, modulate accessibility to glyco sylation enzymes, and enable novel glycan functionalities via multimerization. the igg1 ‐ fc constructs in question were selected from a panel with variable multimerization abilities that depend on the insertion of an igm µ ‐ tailpiece at the c ‐ terminus, the mutation of cysteine residues ( c309l and c575a ), and the insertion of novel n ‐ glyco sylation sites at asn and asn ( see figure 6 ) ( blundell et al. 2017, 2019, 2020; czajkowsky et al. 2015; mekhaiel et al. 2011 ). a monomeric igg ‐ fc with only the classical asn site and a c309l mutation was used as a reference. the remaining five constructs contained a c ‐ terminal µ ‐ tailpiece and a mixture of the following mutations c309l, c575a, d221n, n297a, and n563a, which modulate multimerization and the number of glyco sylation sites. interestingly, the asn site was previously shown to contain hypersialylated n ‐ glycans, which is unusual in igg ‐ fc n ‐ glycans normally found at asn ( blundell et al. 2017 ). these hypersialylated n ‐ glycans enabled strong binding to sialic acid ‐ dependent receptors and were also found to inhibit influenza a and b ‐ induced hemagglutination of human erythrocytes when produced in cho ‐ k1 cells ( blundell et al. 2017, 2019, 2020 ). however, when produced in hek ‐ 293 cells, which express both α2, 3 ‐ and α2, 6 ‐ linked neuac, the constructs were not able to inhibit influenza b ‐ induced hemagglutination, which highlights the importance of cell line ‐ specific glyco sylation capabilities ( blundell et al. 2020 ). while human cells can generate n ‐ glycan structures not seen in cho cells ( e. g., antennal fucosylation or α2, 6 ‐ linked neuac ), the differences between cho ‐ k1 and cho ‐ s cells are more subtle. the results that follow will illustrate the importance of understanding the glyco sylation capabilities of different cho cell lineages in manufacturing biopharmaceuticals. schematic showing the various igg1 ‐ fc glyco sylation mutants produced in cho ‐ k1 and cho ‐ s cells. cys is mutate d to alanine to create the c575a panel of mutants. additionally, cys and leucine are changed to leucine and histidine, respectively, as found in the native igg1 ‐ fc sequence to create the c309l mutants. n ‐ glycans mark the approximate location of the hinge asn, the ch2 asn, and the igm ( μ ) tailpiece asn glyco sylation sites. the multimeric state of the igg1 ‐ fc mutants is shown for igg1 ‐ fc mutants produced in cho ‐ k1 cells ( blundell et al. 2019 ). the n ‐ glyco mic maldi ‐ tof ms spectra of igg1 ‐ fc constructs produced in cho ‐ s cells were obtained and compared to previously reported data from cho ‐ k1 cells ( see figure 7 ). as expected from the constructs produced in cho ‐ k1 cells, cho ‐ s cell constructs containing solely the asn n ‐ glyco sylation site exhibit less processed n ‐ glycans than constructs containing asn and asn. a semi ‐ quantitative analysis illustrates this further, where constructs containing only asn and asn have a higher abundance of galactosylation ( 66. 6 % – 94. 3 % ) and sialylation ( 14. 9 % – 69. 9 % ) than asn ‐ containing constructs ( gal 31. 1 % – 44. 8 %, neuac 0. 4 % – 5. 8 % ) ( see figure 8 ). while the sialylation levels of asn ‐ only constructs from cho ‐ k1 cells are low ( 3. 7 % – 5. 8 % ), sialylation was found to be virtually absent in the same igg1 ‐ fc constructs from cho ‐ s cells ( 0. 4 % ). this can also be observed from the smaller n ‐ glycan mass range, which extends up to m / z 2966 in cho ‐ s cells compared to m / z 4587 in cho ‐ k1 cells for the control c309l construct. the abundance of galactosylated n ‐ glycans was also found to be higher in the asn only construct n563a / c575a when produced in cho ‐ k1 cells ( 43. 5 % vs. 31. 1 % ). interestingly, in the asn ‐ only control fc galactosylation levels were lower in cho ‐ k1 cells ( 37. 5 % ) compared to cho ‐ s cells ( 44. 8 % ). however, when oligomannose n ‐ glycans are excluded from the semi ‐ quantitative analysis, galactosylation levels are largely similar ( 45. 2 % – 45. 3 % ). with respect to the abundance of core ‐ fucosylated n ‐ glycans, no clear pattern was discerned, and there is considerable variability between fc constructs ( 67. 7 % – 95. 5 % ). additionally, the differences in core ‐ fucosylation between cho ‐ k1 and cho ‐ s cells are relatively small. it is noted that while minor neugc ‐ containing structures were identified in low abundances, particularly in hypersialylated constructs, these were not considered for semi ‐ quantitation. maldi ‐ tof mass spectrometry profiles of permethylated n ‐ glycans from human igg1 ‐ fc constructs produced in cho ‐ s and cho ‐ k1 cells. all molecular ions represent the singly charged and sodiated form [ m + na ]. structures shown with brackets have not had their antennal location unequivocally defined. n ‐ glycan compositions of human igg1 ‐ fc constructs produced in cho ‐ k1 and cho ‐ s cells. the different igg ‐ fc constructs are listed as numbers according to the order in figure 6. 1 = control ( c309l ); 2 = n563a _ c575a; 3 = d221n _ c309l _ n297a _ n563a _ c575a; 4 = d221n _ c309l _ n563a _ c575a; 5 = d221n _ c309l _ n297a _ c575a; 6 = d221n _ c575a. the n ‐ glyco sylation sites present in each construct are reiterate d above the top left panel and in the table. overall, four of the six constructs produced in cho ‐ k1 cells have a higher abundance of galactosylated n ‐ glycans, and five have a higher abundance of sialylated n ‐ glycans compared to constructs produced in cho ‐ s cells, which partially aligns with the findings from the host cell n ‐ glyco me experiments reported above. another example of this pattern is the construct containing asn and asn ( d221n / c309l / n297a / c575a ), where galactosylation and sialylation levels were higher in cho ‐ k1 cells ( 94. 3 % gal, 69. 9 % neuac ) relative to cho ‐ s cells ( 77. 0 % gal, 42. 3 % neuac ), despite the higher mass range extending up to m / z 5935 observed in the latter ( it is noted that in the previous study, data were not obtained above m / z 5000 for cho ‐ k1 cells ). however, the asn ‐ only construct ( d221n / c309l / n297a / n563a / c575a ) produced in cho ‐ s cells bucks this trend and shows increased galactosylation ( 88. 4 % vs. 66. 6 % ) and sialylation ( 64. 4 % vs. 14. 9 % ) levels compared to the construct produced in cho ‐ k1 cells. cell line ‐ specific glyco sylation capabilities or natural variation could both explain these opposing patterns.