the impact of increased metabolic pressure from the expression and secretion of recombinant mabs on cho cell n ‐ glyco sylation is not sufficiently understood. in fact, as the cell redirects limited resources toward antibod y production away from biomass production, and the flux in the secretory pathway increases, the expression of glyco syltransferases and glyco sidases, and the golgi residence time of recombinant glyco proteins may be affected ( fan et al. 2015; jimenez del val et al. 2016 ). finally, we analyzed the n ‐ glyco mic profiles of a panel of antibod y ‐ fc constructs with novel glyco sylation sites, produced in cho ‐ s cells, and compared these to previously published data for the same constructs produced in cho ‐ k1 cells. to our knowledge, this is the first study investigating the cellular glyco me of industrially relevant cho cells and the impact of recombinant product expression and secretion on the glyco sylation machinery of cho cells. all cells were grown in cd ‐ cho medium ( gibco, thermofisher scientific, usa ) with the relevant supplements added as shown in table 1. solutions were prewarmed to 37°c and cells were incubated at 37°c with 5 % co2 and orbital shaking ( 16 mm orbit; ssl1 orbital shaker, stuart ) ( see table 1 ). all centrifugations of cho cells were performed at 800 rpm ( 120g; tx ‐ 400 rotor, thermofisher scientific, usa ) for 5 min. during cell revival, a 1 ml cell vial stored in liquid nitrogen ( ln2 ) was thawed and resuspended in 9 ml prewarmed medium. cell density and culture viability were measured using a haemocytometer and trypan blue exclusion, and an nc ‐ 3000 nucleocounter with solution 18 and a8 nc ‐ slides according to manufacturer ' s instructions ( chemometec, denmark ). cells were then seeded at the appropriate density ( see table 1 ) in sterile vented flasks with 50 ml of total culture volume and split every 3 or 4 days. each cell line was cultured in biological triplic ates and cells were harvested after three passages post ‐ revival. cell line ‐ specific incubator growth conditions. l ‐ glutamine ( 8 mm, # 25030081 gibco ) ht supplement ( 10 ml · l, 100× dilution, # 11067030, gibco ) l ‐ glutamine ( 8 mm, # 25030081, gibco ) hygromycin b ( 500 μg · l, # 10687010, gibco ) the construction, expression, and purification of the cho ‐ k1 panel of fc ‐ fragments have been described in detail ( blundell et al. 2019, 2020 ), while the same panel of identical constructs manufactured in cho ‐ s cells is described in baksmeier et al. ( 2021 ). the n ‐ glyco mic analysis is based on a previous protocol ( north et al. 2010 ). on day 4 of the third subculture, 1 × 10 cells were harvested by centrifugation at 800 rpm ( 120g; tx ‐ 400 rotor, thermo ) for 5 min. cell pellets were washed in phosphate ‐ buffered saline and pelleted again prior to storage at −80°c. the cells were then sonicate d in lysis buffer ( 25 mm tris, 150 mm nacl, 5 mm edta, and 1 % chaps, ph 7. 4 ) on ice — 4 × 10 s in continuous mode at 40 amps ( cv188 sonicator, vibra ‐ cell ‐ sonics ) with 1 min on ice in between each sonication step. the samples were then dialyzed against 5 × 4. 5 l of 50 mm ammonium bicarbonate ( ph 8. 5 ) at 4°c for 48 h and then lyophilized. extracted glyco proteins were then reduced with 1 ml of 2 mg / ml dithioth reitol in 0. 6 m tris buffer ( ph 8. 5 ) at 50°c for 60 min. carboxymethylation was then carried out via the addition of 1 ml of 12 mg / ml iodoacetic acid in 0. 6 m tris buffer ( ph 8. 5 ), where samples were incubated at room temperature, in the dark, for 90 min. samples were then dialyzed once more as described above, and lyophilized after 48 h. the reduced and carboxymethylated glyco proteins were resuspended in 700 µl of 50 mm ammonium bicarbonate and tryptically digested with the addition of 50 µl of 10 mg / ml porcine pancreas trypsin ( ec 3. 4. 21. 4; sigma ‐ aldrich ) in 50 mm ammonium bicarbonate buffer ( ph 8. 4 ) at 37°c for 24 h. the reaction was terminated by adding 3 drops of pure acetic acid, and the glyco peptide products were then purified using oasis c18 hlb plus 60 µm cartridges ( waters, usa ) as described previously ( north et al. 2010 ). glyco peptides were resuspended in 200 µl of 50 mm ammonium bicarbonate ( ph 8. 4 ) and 5 µl of recombinant pngase f ( ec 3. 5. 1. 52, roche, switzerland ) in glycerol was added to each sample. the reaction was carried out at 37°c for 24 h — with the addition of a supplementary 5 µl aliquot of pngase f after the first 8 h — and then lyophilized. n ‐ glycans were then resuspended in 200 µl of 5 % acetic acid ( v / v ) and separated from peptides and o ‐ glyco peptides by reverse phase chromatography using sep ‐ pak classic c18 cartridges ( waters, usa ) as described previously ( north et al. 2010 ). n ‐ glycans were eluted in 5 ml of 5 % acetic acid ( v / v ). at this stage, 1 / 3 of the purified n ‐ glycans were aliquoted for enzymatic digestion prior to derivatization, while the remaining 2 / 3 were derivatized directly following lyophilisation. after the aliquots were taken from the 5 ml elution, volumes were reduced on a speedvac, and samples were then lyophilized. the 1 / 3 aliquots of purified n ‐ glycans were resuspended in 400 µl of 50 mm sodium acetate ( ph 5. 5 ) and 8 µl of endo ‐ β ‐ galactosidase ( ec 3. 2. 1. 103, r & d systems, biotechne, usa ) was added to each sample, which were then incubated at 37°c for 24 h, and then lyophilized. digested n ‐ glycans were resuspended in 200 µl of 5 % acetic acid ( v / v ) and purified once more by reverse phase chromatography using sep ‐ pak classic c18 cartridges ( waters, usa ) with the same protocol as the post ‐ pngase f chromatographic step. the samples were then lyophilized and derivatized. n ‐ glycans were permethylated with the sodium hydroxide procedure. first, 5 pellets of sodium hydroxide ( per sample ) were ground in a dry glass mortar with 3 ml of anhydrous dimethylsulfoxide. in total, 1 ml of the resulting slurry was then added to each sample tube together with 0. 5 ml of methyl io dide and mixed vigorously with an automatic shaker for 15 – 45 min ( until the slurry becomes a white paste that no longer mixes ). the reaction was quenched by the drop ‐ wise addition of water with constant agitation, after which 1 ml of chloroform was added to extract the permethylated n ‐ glycans. the mixture was made up to ~ 5 ml with ultra ‐ pure water and vortexed vigorously for 30 s to wash out hydrophilic contaminants. the hydrophobic and hydrophilic layers were separated rapidly by centrifugation, and the aqueous layer was then discarded. a further four washes were performed by adding 5 ml of ultra ‐ pure water, vortexing for 30 s, and discarding the aqueous layer after centrifugation. the chloroform layer was then dried under a gentle stream of nitrogen gas. permethylated n ‐ glycans were further purified from hydrophilic contaminants by reverse phase chromatography using sep ‐ pak classic c18 cartridges ( waters, usa ). the cartridges were conditioned by eluting successively 5 ml methanol, 5 ml ultra ‐ pure water, 5 ml acetonitrile ( mecn ), and re equilibrate d with 3 × 5 ml ultra ‐ pure water. the dry samples from the chloroform extraction were redissolved in 100 µl methanol, after which 100 µl of ultra ‐ pure water was added to each sample prior to loading onto the cartridge. once the sample was loaded, a 5 ml wash with ultra ‐ pure water was eluted and then 3 ml of each 15 %, 35 %, 50 %, and 75 % mecn ( v / v ) were eluted and collected. all fraction volumes were reduced on a speedvac and samples were then lyophilized. the lyophilized permethylated n ‐ glycans were redissolved in 10 µl methanol and a 1 µl aliquot from each fraction was mixed with 1 µl of 3, 4 ‐ diamino ‐ benzophenone ( dabp ) matrix solution ( 10 mg / ml, in 75 % mecn, v / v ).