The effect of ammonia on Chinese hamster ovary (CHO) cell growth and galactosylation of recombinant immunoglobulin (rIgG) was investigated using shaking flasks with serum free media containing 0-15 mM NH(4)Cl. The elevated ammonia inhibited cell growth and negatively affected the galactosylation of rIgG. At 15 mM NH(4)Cl, the proportions of monogalactosylated glycan with fucosex (monogalactosylated glycan with fucose) and digalactosylated glycan with fucose (G2F) were 23.9% and 6.3% lower than those at 0 mM NH(4)Cl, respectively. To reduce ammonia formation by cells, glutamate was examined as a substitute for glutamine. The use of glutamate reduced the accumulation of ammonia and enhanced the production of rIgG while depressing cell growth. At 6 mM glutamate, ammonia level did not exceed 2 mM, which is only one third of that at 6 mM glutamine. Also, a 1.7-fold increase in the titer of rIgG and specific rIgG productivity, q (rIgG), was achieved at 6 mM glutamate. The galactosylation of rIgG was favorable at 6 mM glutamate. The proportion of galactosylated glycans, G1F and G2F, at 6 mM glutamate was 59.8%, but it was 50.4% at 6 mM glutamine. The use of glutamate also increased complement-dependent cytotoxicity activity, one of the effector functions of rIgG. Taken together, substitution of glutamine by glutamate can be considered relevant for the production of rIgG in CHO cells since glutamate not only enhances q (rIgG) but also generates a higher galactosylation essential for the effector function of rIgG.
Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.
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