Download Reduction of metabolic wastes, ammonia and lactate, through the

Reduction of metabolic wastes, ammonia and lactate, through the coupling of glutamine
synthetase selection and lactate dehydrogenase-A down-regulation in CHO cells
Soo Min Noh1, Jin Hyoung Park1, Myung Sin Lim2, Ju Rang Woo2, Jong Won Kim2, Gyun Min Lee 1
1
Department of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea
2
New Drug Development Center, Cheongju 363-951, Republic of Korea
The cultivation of Chinese hamster ovary (CHO) cells for the production of therapeutic
proteins inevitably accompanies the production of metabolic wastes, mostly ammonia and lactate. To
reduce ammonia production, glutamine synthetase (GS) system was used for the expression of
therapeutic monoclonal antibody (mAb) in CHO cells. In addition, the lactate dehydrogenase-A
(LDH-A) gene was down-regulated with shRNA to reduce lactate production. The resulting mAbproducing cell line produced a reduced level of ammonia compared to the host cell line during the
exponential phase due to the over-expression of the GS protein. The down–regulation of the LDH-A
gene in the mAb-producing cell line not only reduced specific lactate production rate but also further
reduced specific ammonia production rate. Waste reduction resulted in the increase of galactosylation
level of the N-glycosylation, improving antibody quality. LDH-A down-regulation was also applied to
the host cell lines of the GS system. However, LDH-A down-regulated host cells could not survive the
pool-selection process in which glutamine was excluded and methionine sulfoximine was added to the
media. Taken together, the application of LDH-A down-regulation in the mAb producing cell line of
the GS system successfully reduced both ammonia and lactate levels, improving the galactosylation of
antibody. However, LDH-A down-regulation could not be applied to the host cell lines because it
hampered the selection process of the GS system.