Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells.

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

Poly-ion complex (PIC) formation of heparin and polyamines: PIC with tetrakis (3-aminopropyl) ammonium allows sustained release of heparin.

Physical mixtures of cationic polymers and heparin have been developed to overcome the limitations of unfractionated heparin. In this study, we found that heparin associates with natural polyamines in water, resulting in the generation of a poly-ion complex (PIC). PIC formation (or stability) was influenced by the concentration and ratio of heparin and polyamines, molecular weight of heparin, nature of polyamines, and pH conditions. Interestingly, the PIC obtained when heparin and tetrakis (3-aminopropyl) ammonium (Taa) were mixed exhibited stability and was sticky in nature. PIC formation was due to an electrostatic interaction between heparin and Taa. Heparin-Taa PIC was administered subcutaneously to mice, and the time to maximum heparin concentration within the therapeutic range of heparin was markedly increased compared to that after a single dose of heparin. These results suggest that the quaternary ammonium structure of Taa is critical for the preparation of a stable PIC, thereby allowing the sustained release of heparin into the blood.